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Abstract:

The invention provides methods for diagnosing, treating, or evaluating
inflammatory and autoimmune diseases by sampling peripheral blood, serum,
plasma, tissue, cerebrospinal fluid, or other bodily fluids from a human
subject having a suspected diagnosis. The sample is analyzed for the
presence and amount of certain cytokines, which provides the diagnosis,
prognosis or evaluation of therapeutic response.

Claims:

1. A method for diagnosing an inflammatory or autoimmune disease state
comprising: (a) obtaining a patient sample; (b) measuring the level of a
plurality of cytokines within the patient sample; (c) comparing cytokine
levels with pre-defined levels of the cytokines found in normal,
inflammatory and/or autoimmune disease states; and (d) determining if a
patient has a given inflammatory or autoimmune disease state based on the
comparison in step (c).

3. The method of claim 1, wherein determining further comprises
classifying the patient as having mild, intermediate, or severe disease.

4. The method of claim 1, wherein the predefined levels comprise
information about a median level of the cytokine found in the patient
sample.

5. The method of claim 4, wherein the patient sample is from a healthy
subject.

6. The method of claim 4, wherein the patient sample is from a diseased
patient.

7. The method of claim 4, wherein the patient sample is from a patient
having major joint destruction and/or extra-articular involvement.

8. The method of claim 1, wherein the disease state is ankylosing
spondylitis and the cytokine is selected from the group consisting of
CCL4, CCL2, CCL11, EGF, IL-1.beta., IL-2, IL-5, IL-6, IL-7, CXCL8, IL-10,
IL-12, IL-13, IL-15, IL-17, TNF-.alpha., IFNγ, GM-CSF, or G-CSF.

9. The method of claim 1, wherein the disease state is psoriatic
arthritis and the cytokine is selected from the group consisting of
GM-CSF, IL-17, IL-2, IL-10, IL-13, IFN-.gamma., IL-6, CCL4/MIP-1.beta.,
CCL11/Eotaxin, EGF, and CCL2/MCP-1.

10. The method of claim 1, wherein the disease state is reactive
arthritis and the cytokine is selected from the group consisting of
IL-12, IFN-.gamma., IL-1.beta., IL-13, IL-17, CCL4/MCP-1, TNF-.alpha.,
IL-4, GM-CSF, CCL11/Eotaxin, EGF, and IL-6.

11. The method of claim 1, wherein the disease state is enteropathic
arthritis and the cytokine is selected from the group consisting of
CXCL8/IL-8, IL-1.beta., IL-4, G-CSF. CCL2/MCP-1, CCL11/Eotaxin, EGF,
IFN-.gamma., and INF-.alpha..

12. The method of claim 1, wherein the disease state is ulcerative
colitis (UC) and the cytokine is selected from the group consisting of
IL-7, CXCL8/IL-8, IFN-.gamma., TNF-.alpha., EGF, VEGF, and IL-1.beta..

13. The method of claim 1, wherein the disease state is Crohn's Disease
(CD) and the cytokine is selected from the group consisting of
TNF-.alpha., IFN-.gamma., IL-1.beta., IL-6, IL-7, IL-13, IL-2, IL-4,
GM-CSF, G-CSF, CCL2/MCP-1, EGF, VEGF, and CXCL8/IL-8.

14. The method of claim 1, wherein the disease state is rheumatoid
arthritis and the cytokine is selected from the group consisting of
IFN-.gamma., IL-1.beta., TNF-.alpha., G-CSF, GM-CSF, IL-6, IL-4, IL-10,
IL-13, IL-5, CCL4/MIP-1.beta., CCL2/MCP-1, EGF, VEGF, and IL-7.

15. The method of claim 1, wherein the disease state is systemic lupus
erythematosus and the cytokine is selected from the group consisting of
IL-10, IL-2, IL-4, IL-6, IFN-.gamma., CCL2/MCP-1, CCL4/MIP-1.beta.,
CXCL8/IL-8, VEGF, EGF, and IL-17.

16. The method of claim 1, wherein the disease state is Familial
Mediterranean Fever (FMF) and the cytokine is selected from the group
consisting of G-CSF, IL-2, IFN-.gamma., TNF-.alpha., IL-1.beta., and
CXCL8/IL-8.

17. The method of claim 1, wherein the disease state is amyotrophic
lateral sclerosis (ALS) and the cytokine is selected from the group
consisting of CCL2/MIP-1.beta., CXCL8/IL-8, IL-12, VEGF, and IL-13.

18. The method of claim 1, wherein the disease state is Irritable Bowel
Syndrome (IBS) and the cytokine is selected from the group consisting of
TNF-.alpha., IFN-.gamma., IL-1.beta., IL-6, IL-7, GM-CSF, G-CSF,
CCL2/MCP-1, and CXCL8/IL-8.

19. The method of claim 1, wherein the disease state is Juvenile
Rheumatoid Arthritis (JRA) and the cytokine is selected from the group
consisting of IFN-.gamma., IL-1.beta., TNF-.alpha., G-CSF, GM-CSF, IL-6,
IL-4, IL-10, IL-13, IL-5, and IL-7.

20. The method of claim 1, wherein the disease state is Sjogren's
Syndrome and the cytokine is selected from the group consisting of
CCL2/MCP-1, IL-12, CXCL8/IL-8, CCL11/Eotaxin, TNFα, IL-2,
IFNα, IL-15, IL17, IL-1.alpha., IL-1.beta., IL-6, and GM-CSF.

21. The method of claim 1, wherein the disease state is early arthritis
and the cytokine is selected from the group consisting of
CCL4/MIP1.beta., CXCL8/IL-8, IL-2, IL-12, IL-17, IL-13, TNFα, IL-4,
IL-5, and IL-10.

22. The method of claim 1, wherein the disease state is neuroinflammation
and the cytokine is selected from the group consisting of CCL2/MCP-1,
IL-12, GM-CSF, G-CSF, M-CSF, IL-6, and IL-17.

23-43. (canceled)

44. A method for determining if a patient with an inflammatory or
autoimmune disease state is predisposed to develop severe inflammatory or
autoimmune disease state, comprising: (a) obtaining a patient sample; (b)
measuring the level of a plurality of cytokines within the patient
sample; (c) comparing cytokine levels with predefined levels of the
cytokines found in patients developing or having severe an inflammatory
or autoimmune disease state; and (d) determining if said patient is
predisposed to develop severe an inflammatory or autoimmune disease
state.

[0006] Many inflammatory, infectious, and autoimmune diseases are now
recognized to involve inflammatory reactions as a major pathologic
feature. Inflammatory processes are driven by cytokine and chemokine
mechanisms. As used herein, the term "cytokine" is defined as any of
several regulatory proteins, such as the interleukins and lymphokines,
that are released by cells of the immune system and act as intercellular
mediators in the generation of an immune response. Cytokines are secreted
by immune or other cells, whose action is on cells of the immune system,
such as, but not limited to, T-cells, B-cells, NK cells and macrophages.
"Chemokines" are defined as chemotactic cytokines produced by a variety
of cell types in acute and chronic inflammation that mobilize and
activate white blood cells. Chemokines can be subdivided into classes on
the basis of the arrangement of a pair of conserved cysteines.

[0007] Cytokines and chemokines are important cell signaling proteins,
mediating a wide range of physiological responses, including immunity,
inflammation, and hematopoiesis. Recently, new biological therapeutic
agents, primarily directed at cytokines, have shown great promise in the
treatment of many inflammatory arthritic diseases. However, despite the
advances in pharmaceutical technology, physicians still prescribe these
expensive, powerful and potentially dangerous therapeutic agents with no
indication of whether patients have the specific inflammatory mediator
antagonized by the pharmaceutical, or whether patients will positively
respond to the medications.

[0008] Despite a number of reports linking the presence or absence of
certain individual cytokines and chemokines to disease states, it is
possible that some assay procedures detect very little cytokine, whereas
others pick up none at all. This difference may be related to the assay
system, to the cytokine, or both. The problem has been reported by the
observations of Cannon et al. (1988), in which the authors showed that
some plasma substance inhibited the assay, affecting detection. The
authors recommend chloroform extraction of plasma to remove interfering
substances, but it is not clear from this study if the plasma factors
simply affect the performance of the assay or are related to the cytokine
itself. This question was further described by Capper et al. (1990),
which showed that IL-1α and IL-1β are bound by proteins and
that the dissociation of these molecules from these serum binding
proteins by acidifying the plasma changes the detectable levels. Thus,
there have been many attempts to measure endogenous cytokines in blood
and other body fluids. However, it is apparent that there is wide
variation in the reported results with regard to cytokine concentration
in the blood and to fluctuations of cytokine concentration in the blood.

[0009] Thus, despite the need to identify cytokine associations with
various inflammatory diseases, there has yet to be established a
definitive link between cytokine expression and diagnosis, prognosis, and
treatment response of such pathologic states.

[0011] The patient sample may comprise peripheral blood, serum, plasma,
cerebrospinal fluid, tissue sample, skin, or other body fluid sample. The
patient may have mild, intermediate, or severe disease. The predefined
levels may comprise information about a median level of the cytokine
found in the patient sample. The patient sample may be from a healthy
subject, a diseased patient or a patient having major joint destruction
and/or extra-articular involvement.

[0015] Where the method disease state is psoriatic arthritis, the cytokine
is selected from the group consisting of GM-CSF, IL-17, IL-2, IL-10,
IL-13, IFN-γ, IL-6, CCL4/MIP-1β, CCL11/Eotaxin, EGF, and
CCL2/MCP-1.

[0016] Where the method disease state is reactive arthritis, the cytokine
is selected from the group consisting of IL-12, IFN-γ, IL-1β,
IL-13, IL-17, CCL4/MCP-1, TNF-α, IL-4, GM-CSF, CCL11/Eotaxin, EGF,
and IL-6.

[0017] Where the method disease state is enteropathic arthritis, the
cytokine is selected from the group consisting of CXCL8/IL-8, IL-1β,
IL-4, G-CSF. CCL2/MCP-1, CCL11/Eotaxin, EGF, IFN-γ, and
TNF-α.

[0018] Where the method disease state is ulcerative colitis (UC), the
cytokine is selected from the group consisting of IL-7, CXCL8/IL-8,
IFN-γ, TNF-α, EGF, VEGF, and IL-1β.

[0019] Where the method disease state is Crohn's Disease (CD), the
cytokine is selected from the group consisting of TNF-α,
IFN-γ, IL-1β, IL-6, IL-7, IL-13, IL-2, IL-4, GM-CSF, G-CSF,
CCL2/MCP-1, EGF, VEGF, and CXCL8/IL-8.

[0020] Where the method disease state is rheumatoid arthritis, the
cytokine is selected from the group consisting of IFN-γ,
IL-1β, TNF-α, G-CSF, GM-CSF, IL-6, IL-4, IL-10, IL-13, IL-5,
CCL4/MIP-1β, CCL2/MCP-1, EGF, VEGF, and IL-7.

[0021] Where the method disease state is systemic lupus erythematosus, the
cytokine is selected from the group consisting of IL-10, IL-2, IL-4,
IL-6, IFN-γ, CCL2/MCP-1, CCL4/MIP-1β, CXCL8/IL-8, VEGF, EGF,
and IL-17.

[0022] Where the method disease state is Familial Mediterranean Fever
(FMF), the cytokine is selected from the group consisting of G-CSF, IL-2,
TNF-α, IL-1β, and CXCL8/IL-8.

[0023] Where the method disease state is amyotrophic lateral sclerosis
(ALS), the cytokine is selected from the group consisting of
CCL2/MIP-1β, CXCL8/IL-8, IL-12, VEGF, and IL-13.

[0024] Where the method disease state is Irritable Bowel Syndrome (IBS),
the cytokine is selected from the group consisting of TNF-α,
IFN-γ, IL-1β, IL-6, IL-7, GM-CSF, G-CSF, CCL2/MCP-1, and
CXCL8/IL-8.

[0025] Where the method disease state is Juvenile Rheumatoid Arthritis
(JRA), the cytokine is selected from the group consisting of IFN-γ,
IL-1β, TNF-α, G-CSF, GM-CSF, IL-6, IL-4, IL-10, IL-13, IL-5,
and IL-7.

[0026] Where the method disease state is Sjogren's Syndrome, the cytokine
is selected from the group consisting of CCL2/MCP-1, IL-12, CXCL8/IL-8,
CCL11/Eotaxin, TNFα, IL-2, IFNα, IL-15, IL17, IL-1α,
IL-1β, IL-6, and GM-CSF.

[0027] Where the method disease state is early arthritis, the cytokine is
selected from the group consisting of CCL4/MIP1β, CXCL8/IL-8, IL-2,
IL-12, IL-17, IL-13, TNFα, IL-4, IL-5, and IL-10.

[0028] Where the method disease state is neuroinflammation, the cytokine
is selected from the group consisting of CCL2/MCP-1, IL-12, GM-CSF,
G-CSF, M-CSF, IL-6, and IL-17.

[0030] Also provided is a method for assessing treatment for an
inflammatory or autoimmune disease state comprising (a) subjecting an
inflammatory or autoimmune disease state patient to a treatment; (b)
obtaining a sample from said patient; (c) measuring the level of a
plurality of cytokines within the patient sample; (d) comparing the level
of a plurality of cytokines with predefined cytokine levels; and (e)
determining if the treatment is efficacious.

[0031] The method may further comprise making a decision regarding
modifying the therapeutic regimen based on said determination of
efficacy. The patient sample may comprise peripheral blood, serum,
plasma, cerebrospinal fluid, tissue sample, skin, or other body fluid
sample. The predefined levels may comprise information about a median
level of the cytokine found in the patient sample. The predefined levels
may comprise pretreatment levels of said one or more cytokines, levels of
said one or more cytokines observed in healthy subjects and/or a patient
with the disease. The patient sample may be from a patient having
extra-articular involvement and/or having major joint destruction.

[0033] Where the method disease state is psoriatic arthritis, the cytokine
is selected from the group consisting of GM-CSF, IL-17, IL-2, IL-10,
IL-13, IFN-γ, IL-6, CCL4/MIP-1β, CCL11/Eotaxin, EGF, and
CCL2/MCP-1.

[0034] Where the method disease state is reactive arthritis, the cytokine
is selected from the group consisting of IL-12, IL-13, IL-17, CCL4/MCP-1,
TNF-α, IL-4, GM-CSF, CCL11/Eotaxin, EGF, and IL-6.

[0035] Where the method disease state is enteropathic arthritis, the
cytokine is selected from the group consisting of CXCL8/IL-8, IL-1β,
IL-4, G-CSF. CCL2/MCP-1, CCL11/Eotaxin, EGF, IFN-γ, and
TNF-α.

[0036] Where the method disease state is ulcerative colitis (UC), the
cytokine is selected from the group consisting of IL-7, CXCL8/IL-8,
TNF-α, EGF, VEGF, and IL-1β.

[0037] Where the method disease state is Crohn's Disease (CD), the
cytokine is selected from the group consisting of TNF-α,
IFN-γ, IL-6, IL-7, IL-13, IL-2, IL-4, GM-CSF, G-CSF, CCL2/MCP-1,
EGF, VEGF, and CXCL8/IL-8.

[0038] Where the method disease state is rheumatoid arthritis, the
cytokine is selected from the group consisting of IFN-γ,
IL-1β, TNF-α, G-CSF, GM-CSF, IL-6, IL-4, IL-10, IL-13, IL-5,
CCL4/MIP-1β, CCL2/MCP-1, EGF, VEGF, and IL-7.

[0039] Where the method disease state is systemic lupus erythematosus, the
cytokine is selected from the group consisting of IL-10, IL-2, IL-4,
IL-6, IFN-γ, CCL2/MCP-1, CCL4/MIP-1β, CXCL8/IL-8, VEGF, EGF,
and IL-17.

[0040] Where the method disease state is Familial Mediterranean Fever
(FMF), the cytokine is selected from the group consisting of G-CSF, IL-2,
IFN-γ, TNF-α, IL-1β, and CXCL8/IL-8.

[0041] Where the method disease state is amyotrophic lateral sclerosis
(ALS), the cytokine is selected from the group consisting of
CCL2/MIP-1β, CXCL8/IL-8, IL-12, VEGF, and IL-13.

[0042] Where the method disease state is Irritable Bowel Syndrome (IBS),
the cytokine is selected from the group consisting of TNF-α,
IFN-γ, IL-1β, IL-6, GM-CSF, G-CSF, CCL2/MCP-1, and CXCL8/IL-8.

[0043] Where the method disease state is Juvenile Rheumatoid Arthritis
(JRA), the cytokine is selected from the group consisting of IFN-γ,
IL-1β, TNF-α, G-CSF, GM-CSF, IL-6, IL-4, IL-10, IL-13, IL-5,
and IL-7.

[0044] Where the method disease state is Sjogren's Syndrome, the cytokine
is selected from the group consisting of CCL2/MCP-1, IL-12, CXCL8/IL-8,
CCL11/Eotaxin, TNFα, IL-2, IFNα, IL17, IL-1α,
IL-1β, IL-6, and GM-CSF.

[0045] Where the method disease state is early arthritis, the cytokine is
selected from the group consisting of CCL4/MIP1β, CXCL8/IL-8, IL-2,
IL-12, IL-17, IL-13, TNFα, IL-4, IL-5, and IL-10.

[0046] Where the method disease state is neuroinflammation, the cytokine
is selected from the group consisting of CCL2/MCP-1, IL-12, GM-CSF,
G-CSF, M-CSF, IL-6, and IL-17.

[0048] In yet another embodiment, there is provided a method for
determining if a patient with an inflammatory or autoimmune disease state
is predisposed to develop severe inflammatory or autoimmune disease
state, comprising (a) obtaining a patient sample; (b) measuring the level
of a plurality of cytokines within the patient sample; (c) comparing
cytokine levels with predefined levels of the cytokines found in patients
developing or having severe an inflammatory or autoimmune disease state;
and (d) determining if said patient is predisposed to develop severe an
inflammatory or autoimmune disease state.

[0049] The patient sample may comprise peripheral blood, serum, plasma,
cerebrospinal fluid, tissue sample, or other body fluid sample. The
predefined levels may comprise information about a median level of the
cytokine found in the patient sample. The method may further comprise
obtaining a plurality of patient symptoms. The patient sample may be from
a patient having extra-articular involvement and/or having major joint
destruction.

[0051] Where the method disease state is psoriatic arthritis, the cytokine
is selected from the group consisting of GM-CSF, IL-17, IL-2, IL-10,
IL-13, IFN-γ, CCL4/MIP-1β, CCL11/Eotaxin, EGF, and CCL2/MCP-1.

[0052] Where the method disease state is reactive arthritis, the cytokine
is selected from the group consisting of IL-12, IFN-γ, IL-1β,
IL-13, IL-17, CCL4/MCP-1, TNF-α, IL-4, GM-CSF, CCL11/Eotaxin, EGF,
and IL-6.

[0053] Where the method disease state is enteropathic arthritis, the
cytokine is selected from the group consisting of CXCL8/IL-8, IL-1β,
IL-4, G-CSF. CCL2/MCP-1, CCL11/Eotaxin, EGF, IFN-γ, and
TNF-α.

[0054] Where the method disease state is ulcerative colitis (UC), the
cytokine is selected from the group consisting of IL-7, CXCL8/IL-8,
IFN-γ, TNF-α, EGF, VEGF, and IL-1β.

[0055] Where the method disease state is Crohn's Disease (CD), the
cytokine is selected from the group consisting of TNF-α,
IFN-γ, IL-1β, IL-6, IL-7, IL-13, IL-2, IL-4, GM-CSF, G-CSF,
CCL2/MCP-1, EGF, VEGF, and CXCL8/IL-8.

[0056] Where the method disease state is rheumatoid arthritis, the
cytokine is selected from the group consisting of IFN-γ,
TNF-α, G-CSF, GM-CSF, IL-6, IL-4, IL-10, IL-13, IL-5,
CCL4/MIP-1β, CCL2/MCP-1, EGF, VEGF, and IL-7.

[0057] Where the method disease state is systemic lupus erythematosus, the
cytokine is selected from the group consisting of IL-10, IL-2, IL-4,
IL-6, IFN-γ, CCL2/MCP-1, CCL4/MIP-1β, CXCL8/IL-8, VEGF, EGF,
and IL-17.

[0058] Where the method disease state is Familial Mediterranean Fever
(FMF), the cytokine is selected from the group consisting of G-CSF, IL-2,
IFN-γ, TNF-α, IL-1β, and CXCL8/IL-8.

[0059] Where the method disease state is amyotrophic lateral sclerosis
(ALS), the cytokine is selected from the group consisting of
CCL2/MIP-1β, CXCL8/IL-8, IL-12, VEGF, and IL-13.

[0060] Where the method disease state is Irritable Bowel Syndrome (IBS),
the cytokine is selected from the group consisting of TNF-α,
IFN-γ, IL-1β, IL-6, IL-7, GM-CSF, G-CSF, CCL2/MCP-1, and
CXCL8/IL-8. Where the method disease state is Juvenile Rheumatoid
Arthritis (JRA), the cytokine is selected from the group consisting of
IFN-γ, IL-1 TNF-α, G-CSF, GM-CSF, IL-6, IL-4, IL-10, IL-13,
IL-5, and IL-7.

[0061] Where the method disease state is Sjogren's Syndrome, the cytokine
is selected from the group consisting of CCL2/MCP-1, IL-12, CXCL8/IL-8,
CCL11/Eotaxin, TNFα, IL-2, IFNα, IL-15, IL17, IL-1α,
IL-1β, IL-6, and GM-CSF.

[0062] Where the method disease state is early arthritis, the cytokine is
selected from the group consisting of CCL4/MIP1β, CXCL8/IL-8, IL-2,
IL-12, IL-17, IL-13, TNFα, IL-4, IL-5, and IL-10.

[0063] Where the method disease state is neuroinflammation, the cytokine
is selected from the group consisting of CCL2/MCP-1, IL-12, GM-CSF,
G-CSF, M-CSF, IL-6, and IL-17.

[0065] Also provided are kits: [0066] for providing diagnostic
information about ankylosing spondylitis comprising a nucleic acid or
antibody probe for determining the cytokine level of two or more of the
cytokines selected from the group consisting of CCL4, CCL2, CCL11, EGF,
IL-1β, IL-2, IL-5, IL-6, IL-7, CXCL8, IL-10, IL-12, IL-13, IL-15,
IL-17, TNF-α, GM-CSF, or G-CSF; for providing diagnostic
information about an psoriatic arthritis comprising a nucleic acid or
antibody probe for determining the cytokine level of two or more of the
cytokines selected from the group consisting of GM-CSF, IL-17, IL-2,
IL-10, IL-13, IFN-γ, IL-6, CCL4/MIP-1β, CCL11/Eotaxin, EGF,
and CCL2/MCP-1; [0067] for providing diagnostic information about
reactive arthritis comprising a nucleic acid or antibody probe for
determining the cytokine level of two or more of the cytokines selected
from the group consisting of IL-12, IFN-γ, IL-1β, IL-13,
IL-17, CCL4/MCP-1, TNF-α, IL-4, GM-CSF, CCL11/Eotaxin, EGF, and
IL-6; [0068] for providing diagnostic information about enteropathic
arthritis comprising a nucleic acid or antibody probe for determining the
cytokine level of two or more of the cytokines selected from the group
consisting of CXCL8/IL-8, IL-1β, IL-4, G-CSF, CCL2/MCP-1,
CCL11/Eotaxin, EGF, IFN-γ, and TNF-β; [0069] for providing
diagnostic information about an ulcerative colitis comprising a nucleic
acid or antibody probe for determining the cytokine level of two or more
of the cytokines selected from the group consisting of IL-7, CXCL8/IL-8,
IFN-γ, TNF-α, EGF, VEGF, and IL-1β; [0070] for providing
diagnostic information about Crohn's disease comprising a nucleic acid or
antibody probe for determining the cytokine level of two or more of the
cytokines selected from the group consisting of TNF-α, IFN-γ,
IL-1β, IL-6, IL-7, IL-13, IL-2, IL-4, GM-CSF, G-CSF, CCL2/MCP-1,
EGF, VEGF, and CXCL8/IL-8; [0071] for providing diagnostic information
about rheumatoid arthritis comprising a nucleic acid or antibody probe
for determining the cytokine level of two or more of the cytokines
selected from the group consisting of IFN-γ, IL-1β,
TNF-α, G-CSF, GM-CSF, IL-6, IL-10, IL-13, IL-5, CCL4/MIP-1β,
CCL2/MCP-1, EGF, VEGF, and IL-7; [0072] for providing diagnostic
information about systemic lupus erythematosus comprising a nucleic acid
or antibody probe for determining the cytokine level of two or more of
the cytokines selected from the group consisting of IL-10, IL-2, IL-4,
IL-6, IFN-γ, CCL2/MCP-1, CCL4/MIP-1β, CXCL8/IL-8, VEGF, EGF,
and IL-17; [0073] for providing diagnostic information about Familial
Mediterranean Fever comprising a nucleic acid or antibody probe for
determining the cytokine level of two or more of the cytokines selected
from the group consisting of G-CSF, IL-2, IFN-γ, TNF-α,
IL-1β, and CXCL8/IL-8; [0074] for providing diagnostic information
about amyotrophic lateral sclerosis (ALS) comprising a nucleic acid or
antibody probe for determining the cytokine level of two or more of the
cytokines selected from the group consisting of CCL2/MIP-1β,
CXCL8/IL-8, IL-12, VEGF, and IL-13; [0075] for providing diagnostic
information about Irritable Bowel Syndrome (IBS) comprising a nucleic
acid or antibody probe for determining the cytokine level of two or more
of the cytokines selected from the group consisting of TNF-α,
IFN-γ, IL-6, IL-7, GM-CSF, G-CSF, CCL2/MCP-1, and CXCL8/IL-8;
[0076] for providing diagnostic information about Juvenile Rheumatoid
Arthritis (JRA) comprising a nucleic acid or antibody probe for
determining the cytokine level of two or more of the cytokines selected
from the group consisting of IFN-γ, IL-1β, TNF-α, G-CSF,
GM-CSF, IL-6, IL-4, IL-10, IL-13, IL-5, and IL-7; [0077] for providing
diagnostic information about Sjogren's Syndrome comprising a nucleic acid
or antibody probe for determining the cytokine level of two or more of
the cytokines selected from the group consisting of CCL2/MCP-1, IL-12,
CXCL8/IL-8, CCL11/Eotaxin, TNFα, IL-2, IFNα, IL-15, IL17,
IL-1α, IL-1β, IL-6, and GM-CSF; [0078] for providing
diagnostic information about early arthritis comprising a nucleic acid or
antibody probe for determining the cytokine level of two or more of the
cytokines selected from the group consisting of CCL4/MIP1β,
CXCL8/IL-8, IL-2, IL-12, IL-17, IL-13, TNFα, IL-4, IL-5, and IL-10;
[0079] for providing diagnostic information about psoriasis comprising a
nucleic acid or antibody probe for determining the cytokine level of two
or more of the cytokines selected from the group consisting of IL-6,
IL-10, IL-2, IL-4, IFN-γ, CCL2/MCP-1, and IL-17; [0080] for
providing diagnostic information about neuroinflammation comprising a
nucleic acid or antibody probe for determining the cytokine level of two
or more of the cytokines selected from the group consisting of
CCL2/MCP-1, IL-12, GM-CSF, G-CSF, M-CSF, IL-6, and IL-17;

[0082] As used herein, "a" or "an" may mean one or more. As used herein in
the claim(s), when used in conjunction with the word "comprising," the
words "a" or "an" may mean one or more than one. As used herein "another"
may mean at least a second or more.

[0083] These, and other, embodiments of the invention will be better
appreciated and understood when considered in conjunction with the
following description and the accompanying drawings. It should be
understood, however, that the following description, while indicating
various embodiments of the invention and numerous specific details
thereof, is given by way of illustration and not of limitation. Many
substitutions, modifications, additions and/or rearrangements may be made
within the scope of the invention without departing from the spirit
thereof, and the invention includes all such substitutions,
modifications, additions and/or rearrangements.

BRIEF DESCRIPTION OF THE DRAWINGS

[0084] The drawings accompanying and forming part of this specification
are included to depict certain aspects of the invention. A clearer
conception of the invention, and of the components and operation of
systems provided with the invention, will become more readily apparent by
referring to the exemplary, and therefore non-limiting, embodiments
illustrated in the drawings, wherein like reference numerals (if they
occur in more than one view) designate the same elements. The invention
may be better understood by reference to one or more of these drawings in
combination with the description presented herein. It should be noted
that the features illustrated in the drawings are not necessarily drawn
to scale.

[0089] FIG. 5--Stepwise Discriminate Function Analysis Graph Showing
Clustering of Normal Controls (red), HLA B27 positive Individuals (grey),
and AS patients (blue/green). Roots used as coordinates were calculated
as described in Table 1 legend. Note that the IRA B27 positive
individuals (grey) appear to form a link between the normal controls
(red) and clinically diagnosed AS patients (blue/green), once again
confirming the importance of HLA B27 positive in disease development. It
may also be of clinical interest to note that a few of the HLA B27
positive healthy individuals presented some early symptoms of AS, but
those symptoms were insufficient to qualify them for a diagnosis of AS
under the Bath criteria. These data indicate that an earlier disease
diagnosis may be warranted in those cases.

[0103] FIG. 19-Distribution of IL-13/IL-12 ratios in CSF from ALS and
non-ALS subjects.

[0104] FIG. 20--Cytokine Profile of Sjogren's Syndrome Patients.

[0105] FIG. 21--Cytokine Profile of Early Arthritis Patients.

[0106] FIG. 22--Cytokine Profile of Psoriasis Patients.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0107] The present invention is premised on the inventors' hypothesis that
examining cytokine expression profiles will prove both diagnostic and
prognostic for various disease states as well as predict and evaluate
treatment response. By applying this hypothesis to a variety of disease
states, the inventors determined the relevant cytokine profiles for the
presence of various disease states, for the propensity of subjects to
develop such disease states, and for the prediction and evaluation of
treatment response in subjects treated with various pharmaceutical or
biological agents. While some cytokines may have previously been
associated with a disease state on an individual basis, this is the first
time that expression profiling, as applied particularly to cytokines, has
been used to diagnose, prognose, predict and evaluate treatment response
in disease.

[0108] The invention and the various features and advantageous details
thereof are explained more fully with reference to the non-limiting
embodiments that are illustrated in the accompanying drawings and
detailed in the following description. Descriptions of well known
starting materials, processing techniques, components and equipment are
omitted so as not to unnecessarily obscure the invention in detail. It
should be understood, however, that the detailed description and the
specific examples, while indicating preferred embodiments of the
invention, are given by way of illustration only and not by way of
limitation. Various substitutions, modifications, additions and/or
rearrangements within the spirit and/or scope of the underlying inventive
concept will become apparent to those skilled in the art from this
disclosure.

I. CYTOKINE INVOLVEMENT IN DISEASE

[0109] Interleukin-1α (IL-1α) is expressed in large amounts by
human keratinocytes. IL-1-α is also produced also by activated
macrophages from different sources (alveolar macrophages, Kupffer cells,
adherent spleen and peritoneal macrophages) and also by peripheral
neutrophil granulocytes, endothelial cells, fibroblasts, smooth muscle
cells, keratinocytes, Langerhans cells of the skin, osteoclasts,
astrocytes, epithelial cells of the thymus and the cornea, T-cells, and
B-cells.

[0110] The concentrations of IL-1 observed in the cerebrospinal fluid are
due to local synthesis and also due to the direct transport of IL-1
through the blood-brain barrier by means of a saturable carrier system
and the ability of activated T-lymphocytes to pass this barrier.
Interleukin-1β (IL-1β) is constitutively expressed in the
brain.

[0111] IL-1α and IL-1β are biologically more or less equivalent
pleiotropic factors that act locally and also systemically. Only a few
functional differences between the factors have been described.

[0112] Some of the biological activities of IL-1 are mediated indirectly
by the induction of the synthesis of other mediators including ACTH
(Corticotropin), PGE2, PF4 (platelet factor-4), CSF (colony stimulating
factors), IL-6, and CXCL8 (IL-8).

[0113] The synthesis of IL-1 can be induced by other cytokines including
TNF-α, IFN-α. IFN-γ, and IFN-β and also by
bacterial endotoxins, viruses, mitogens, and antigens. In human skin
fibroblasts, IL-1α and TNF-α induce the synthesis of
IL-1β. Human mononuclear cells are very sensitive to bacterial
endotoxins and synthesize IL-1 in response to picogram/mL amounts of
endotoxins. In human monocytes bacterial lipopolysaccharides induce
approximately tenfold more mRNA and the respective proteins for
IL-1β than for IL-1α.

The synthesis of IL-1 is controlled by a complex feedback loop because
IL-1 is capable also of inhibiting or promoting its own synthesis,
depending on conditions and cell types.

[0114] The main biological activity of IL-1 is the stimulation of T-helper
cells, which are induced to secrete IL-2 and to express IL-2 receptors.
Virus-infected macrophages produce large amounts of an IL-1 inhibitor
(IL-1ra) that may support opportunistic infections and transformation of
cells in patients with T-cell maturation defects.

[0115] IL-1 acts directly on B-cells, promoting their proliferation and
the synthesis of immunoglobulins. IL-1 also functions as one of the
priming factor that makes B-cells responsive to IL-5. IL-1 stimulates the
proliferation and activation of NK-cells and fibroblasts, thymocytes,
glioblastoma cells. It also promotes the proliferation of astroglia and
microglia and may be involved in pathological processes such as
astrogliosis and demyelination. The IL-1 mediated proliferation of
lymphocytes is inhibited by TGF-β-1 and TGF-β-2.

[0116] A mechanism of autocrine growth control by IL-1 has been suggested
for leukemic blast cells in which the uncontrolled synthesis of IL-1 is
thought to lead to the production of colony stimulating factors (CSF)
that in turn promote the proliferation of these cells. In combination
with other cytokines, IL-1 appears to be an autocrine growth modulator
for human gastric and thyroid carcinoma cells. The growth-promoting
activities of IL-1 are mediated indirectly in some systems by regulating
the expression of high affinity receptors for another cytokine, FGF. IL-1
has been shown also to be radioprotective.

[0117] IL-1 also has antiproliferative and cytocidal activities on certain
tumor cell types. It supports the monocyte mediated tumor cytotoxicity
and induces tumor regression. IL-1 is cytotoxic for insulin-producing
beta cells of the Langerhans islets of the pancreas.

IL-1 inhibits the growth of endothelial cells in vivo and in vitro. IL-1
causes many alterations of endothelial functions in vivo. It promotes
thrombotic processes and attenuates anticoagulatory mechanisms. IL-1
therefore plays an important role in pathological processes such as
venous thrombosis, arteriosclerosis, vasculitis, and disseminated
intravasal coagulation.

[0118] IL-1 promotes the adhesion of neutrophils, monocytes, T-cells, and
B-cells by enhancing the expression of adhesion molecules such as CAM-1
(intercellular adhesion molecule) and ELAM (endothelial leukocyte
adhesion molecule). The expression of membrane-associated thrombomodulin
is decreased by IL-1.

[0119] IL-1 also influences the functional activities of Langerhans cells
of the skin. These cells are not capable of eliciting primary immune
responses (for example, contact sensibilisation). IL-1 (and also GM-CSF)
convert these cells into potent immunostimulatory dendritic cells. The
Langerhans cells therefore constitute an in situ reservoir for
immunologically immature lymphoid dendritic cells. The increased ability
of maturated Langerhans cell to process antigens is decreased by
TNF-α

[0120] IL-1 in combination with other cytokines is an important mediator
of inflammatory reactions. IL-1 markedly enhances the metabolism of
arachidonic acid (in particular of prostacyclin and PGE2) in inflammatory
cells such as fibroblasts, synovial cells, chondrocytes, endothelial
cells, hepatocytes, and osteoclasts. In addition one observes an
increased secretion of inflammatory proteins such as neutral proteases
(collagenase, elastase and plasminogen activator). This activity of IL-1
antagonizes the effects of TGF-β on the extracellular matrix.

[0121] IL-1 is also a strong chemoattractant for leukocytes. In vivo the
injection of IL-1 leads to the local accumulation of neutrophils at the
site of injection. IL-1 also activates oxidative metabolism in
neutrophils.

[0122] In combination with TNF, IL-1 appears to be involved in the
generation of lytic bone lesions. IL-1 activates osteoclasts and
therefore suppresses the formation of new bone. Low concentrations of
IL-1, however, promote new bone growth. IL-1 inhibits the enzyme
lipoprotein lipase in adipocytes. In vascular smooth muscle cells and
skin fibroblasts IL-1 induces the synthesis of bFGF, which is a mitogen
for these cells.

[0123] Like Interleukin-2 (IL-2), IL-1 also modulates the
electrophysiological behavior of neurons. IL-1 also directly affects the
central nervous system as an afferent signal modulating the release of a
number of hormones and activates the
hypothalamic-pituitary-adrenocortical (HPA) axis. IL-1 activates the
serotoninergic system. IL-1 also functions as an endogenous pyrogen and
induces a significant elevation of body temperature by causing the
release of prostaglandins in the thermoregulatory center of the
hypothalamus. This activity is inhibited by Alpha-Melanocyte stimulating
hormone (alpha-MSH). IL-1, like IL6, stimulates the synthesis of ACTH
(corticotropin) in the pituitary. Glucocorticosteroids on the other hand
increase the expression of IL-1 receptors. In blood serum IL-1 has been
shown to decrease plasma concentrations of iron and zinc. In Schwann
cells and fibroblast-like cells of nervous tissue IL-1 induces the
synthesis of NGF.

[0124] IL-1β immunoreactive nerve fibers in human hypothalamus
innervate those endocrine and autonomous nuclei controlling central
aspects of acute phase reaction. Astrocytes proliferate in the presence
of IL-1 and release IL-3, which is a growth factor for microglia and
peritoneal macrophages. In Astrocytes IL-1 also promotes the synthesis of
GM-CSF, IL-6 and TNF. In the central nervous system IL-1 is involved also
in the induction of the so-called slow-wave sleep. IL-1β can prevent
the depression of antibody responses observed during sleep deprivation.

[0125] IL-1 synergises with some other factors. The effect of IL-1 on the
proliferation of thymocytes involves the stimulation of IL-2 synthesis
and the expression of IL-2 receptors on T-cells. For certain T-helper
cell populations and also for B-cells IL-1 functions as an additional
growth factor. IL-1 potentiates the effects of colony stimulating factors
(CSFs) and promotes the generation of myeloid progenitor cells from stem
cells. IL-1 synergises with GM-CSF in the induction of macrophage colony
growth. IL-1 can also induce the synthesis of G-CSF and M-CSF by bone
marrow stromal cells and stimulates the synthesis of GM-CSF and G-CSF by
human skin cells and of GM-CSF by peripheral blood lymphocytes. By
enhancing the expression of receptors for colony stimulating factors IL-1
is involved in various processes of hematopoiesis. IL-1 also induces the
proliferation of pluripotent bone marrow progenitor cells.

[0126] Peter et al., (1991) examined interleukin-1β (IL-1β) and
tumor necrosis factor (TNF) in cerebrospinal fluid (CSF) and serum of
multiple sclerosis patients and normal controls. They concluded that the
levels of these cytokines, in both fluids, were not of prognostic or
diagnostic utility. Westacott et al. (1990) used immunoassays to measure
cytokines in synovial fluid of patients with rheumatic disease. A factor
with IL-1-like bioactivity was detected in the gingival fluid of
clinically normal humans (Oppenheim et al., 1982), the activity being
higher in inflamed than non-inflamed gingival regions. The gingival fluid
factor exhibited molecular weights corresponding to both IL-1 and
epidermal thymocyte-activating factor (Charon et al., 1982). Studies by
Jandinski and colleagues (Jandinski et al., 1988a; 1988b; 1988c) reported
the presence of IL-1β in periodontal tissue, while IL-1 predominated
in gingival crevicular fluid of patients with periodontal disease.
Another study (Kabashimi et al., 1990) utilizing polyclonal antisera to
recombinant human IL-1α and IL-1β and measurement by Western
blotting, disclosed that the majority of the IL-1 bioactivity found in
gingival crevicular fluid of patients with chronic inflammatory
periodontal disease was IL-1, generally considered the membrane-bound
form of IL-1. The suggestion was made that the IL-1 was derived by
enzymatic cleavage from the cell surface. In this latter study, special
care was taken to avoid contamination of the gingival fluid with saliva.
These facts argue strongly against a salivary origin for the gingival
fluid IL-1.

[0127] Under physiological conditions, interleukin-2 (IL-2) is produced
mainly by T-cells expressing the surface antigen CD4 following cell
activation by mitogens or allogens. Resting cells do not produce IL-2
(Smith, 1988). Transformed T-cells and B-cells, leukemia cells, LAK cells
(lymphocyte-activated killer cells) and NK-cells also secrete IL-2. IL-2
displays significant anti-tumor activity for a variety of tumor cell
types because it supports the proliferation and clonal expansion of
T-cells that specifically attack certain tumor types (Williams et al.,
1991). IL-2 is increasingly used to treat patients with cancers
refractory to conventional treatment (Waldman et al., 1993). Objective
and long-lived clinical responses have been documented also in a
proportion of patients with melanoma or acute myeloid leukemia (Broom et
al., 1992).

[0128] Interleukin-4 (IL-4) is produced mainly by a subpopulation of
activated T-cells (Th2), which are the biologically most active helper
cells for B-cells and which also secrete IL-5 and IL-6. Another
subpopulation of T-cells (Th1) also produces IL-4, but to a lesser
extent. Non-T/Non-B-cells of the mast cell lineage also produce IL-4
(Boulay et al., 1992). IL-4 promotes the proliferation and
differentiation of activated B-cells, the expression of class II MHC
antigens, and of low affinity IgE receptors in resting B-cells (Jansen et
al., 1990). IL-4 is thought to be an autocrine growth modulator for
Hodgkin's lymphomas (Okabe et al., 1992). IL-4 enhances expression of
class II MHC antigens on B-cells. It can promote their capacity to
respond to other B-cell stimuli and to present antigens for T-cells (Paul
et al., 1991). This may be one way to promote the clonal expansion of
specific B-cells and the immune system may thus be able to respond to
very low concentrations of antigens. The production of IL-4 by Non-B
Non-T-cells is stimulated if these cells interact with other cells via
their Fc receptors for IgE or IgG (Callard et al., 1991) This effect can
be enhanced by IL-3. IL-4 also inhibits cell activation of NK-cells
induced by IL-2 (Paul et al., 1987).

[0129] IL-4 may be of clinical importance in the treatment of inflammatory
diseases and autoimmune diseases since it inhibits the production of
inflammatory cytokines such as IL-1, IL-6, and TNF-α by monocytes
and T-cells (Dullens et al., 1992). IL-4 may be useful also in the
treatment of solid tumors, of hematopoietic systemic diseases, and of
immune defects. IL4 inhibits the growth of colon and mammary carcinomas
(Toi et al., 1992). It has been shown to augment the development of
lymphokine-activated killer cells (LAK cells). IL-4 may play an essential
role in the pathogenesis of chronic lymphocytic leukemia disease, which
is characterized by the accumulation of slow-dividing and long-lived
monoclonal B-cells arrested at the intermediate stage of their
differentiation by preventing both the death and the proliferation of the
malignant B-cells. It protects chronic lymphocytic leukemic B-cells from
death by apoptosis and upregulates the expression of a protective gene,
BCL-2 (Dancescu et al., 1992).

[0130] Interleukin-5 (IL-5) is produced by T-cells. IL-5 is a specific
hematopoietic growth factor that is responsible for the growth and
differentiation of eosinophils (Takatsu et al., 1992). IL-5 strongly
stimulates the proliferation, cell activation, and differentiation of
eosinophilic granulocytes. B-cells can be made responsive to IL-5 by
treatment with suboptimal doses of IL-1. IL-5 also promotes the
generation of cytotoxic T-cells from thymocytes (Sanderson et al., 1988).
A possible clinical application is suggested by the activity of IL-5 on
eosinophils. Animal experiments have shown that eosinophilia elicited by
nematode infections in mice and the concomitant infiltration of the lung
with eosinophils can be prevented by administration of monoclonal animals
directed against IL-5 (Coffman et al., 1989).

[0132] The determination of IL-6 serum levels may be useful to monitor the
activity of myelomas and to calculate tumor cell masses; Low IL-6 serum
levels are observed in monoclonal gammopathies and in smoldering myelomas
while IL-6 serum levels are markedly increased in patients with
progressive disease and also in patients with plasma cell leukemia (Van
Oers et al., 1993). A blockade of the IL-6 receptor or the inhibition of
IL-6 by monoclonal antibodies may be a way to delay or prevent the
maturation of B-cells into plasma cells.

[0133] The deregulated expression of IL-6 is probably one of the major
factor involved in the pathogenesis of a number of diseases (Leger-Ravet
et al., 1991). The excessive overproduction of IL-6 has been observed in
various pathological conditions such as rheumatoid arthritis, multiple
myeloma, Lennert syndrome (histiocytic lymphoma), Castleman's disease
(lymphadenopathy with massive infiltration of plasma cells, hyper
gamma-globulinemia, anemia, and enhanced concentrations of acute phase
proteins), cardiac myxomas and liver cirrhosis (Hsu et al., 1993). The
constitutive synthesis of IL-6 by glioblastomas and the secretion of IL-6
into the cerebrospinal fluid may explain the elevated levels of acute
phase proteins and immune complexes in the serum (Mule et al. 1991).

[0134] IL-6 probably also plays a role in the pathogenesis of chronic
polyarthritis because excessive concentrations of IL-6 are found in the
synovial fluid. It has been suggested that IL-6, due to its effects on
hematopoietic cells, may be suitable for the treatment of certain types
of anemia and thrombocytopenia (Brach et al., 1992). Pretreatment with
IL-3 and subsequent administration of IL-6 has been shown to increase
platelet counts. In combination with other cytokines (for example, IL-2)
IL-6 may be useful in the treatment of some tumor types (Duliens et a.l,
1991).

[0135] Very high levels of IL-6 in the cerebrospinal fluid are observed
frequently in bacterial and viral meningitis. The detection of elevated
concentrations of IL-6 in the urine of transplanted patients may be an
early indicator of a graft-versus-host reaction (Kishimoto et al., 1990).
The detection of IL-6 in the amniotic fluid may be an indication of
intra-amniotic infections. In inflammatory intestinal diseases elevated
plasma levels of IL-6 may be an indicator of disease status (Wolvekamp et
al., 1990). In patients with mesangioproliferative glomerulonephritis
elevated urine levels of IL-6 are also an indicator of disease status
(Van Snick et al., 1990). Monitoring of postoperative serum IL-6 levels
may be more helpful than monitoring of C-reactive protein levels for
estimation of inflammatory status and early detection of an acute phase
reaction (Ohzato et al., 1992). Serum and urinary IL-6 levels have been
shown to be predicting factors of Kawasaki disease activity (Furukawa et
al., 1992).

[0136] Interleukin-7. (IL-7) is secreted constitutively into the
conditioned medium of adherent bone marrow stromal cells and thymic
cells. Murine and human keratinocytes have been shown also to express and
secrete IL-7. IL-7 may be of clinical significance for adoptive
immunotherapy because it is capable in vivo to cause the
CD4(+)T-cell-dependent destruction of tumor cells (Hock et al., 1991).
IL-7 has been shown also to induce LAK cells activity comparable
quantitatively to that induced by IL-2 in cells obtained from patients
early after autologous or syngeneic bone marrow transplantation (Pavletic
et al., 1993). It induces an even greater LAK cell activity in vitro in
peripheral blood mononuclear cells obtained after autologous bone marrow
transplantation and preactivated in vivo by IL-2 therapy (Stotter et al.,
1991). It has therefore been suggested that IL-7, alone or in combination
with IL-2, may be used as a consolidative immunotherapy for malignancies
in patients after autologous bone marrow transplantation.

[0137] Participation of IL-7 in the pathogenesis of inflammatory skin
diseases and cutaneous T-cell lymphomas is suggested by the
growth-promoting effects of IL-7 and its synthesis by keratinocytes. IL-7
may contribute to disturbances of immune regulatory T-cells in ulcerative
colitis because a serum factor from patients with ulcerative colitis that
induces proliferation of intrathymic T-cells has been found to be
identical with IL-7 (Watanabe et al., 1997).

[0138] Interleukin-8 (IL-8), now called CXCL8, is produced by stimulated
monocytes but not by tissue macrophages and T-lymphocytes. CXCL8 is
produced also by macrophages, fibroblasts, endothelial cells,
keratinocytes, melanocytes, hepatocytes, chondrocytes, and a number of
tumor cell line (Koch et al., 1991). In many cell types the synthesis of
CXCL8 is strongly stimulated by IL-1 and TNF-α. The synthesis of
CXCL8 is induced also by phytohemagglutinins, concanavalin A,
double-stranded RNA, phrobol esters, sodium urate crystals, viruses, and
bacterial lipopolysaccharides (Zwahlen et al., 1993). The expression of
CXCL8 from resting and stimulated human blood monocytes is up-regulated
by IL-7. In chondrocytes the synthesis of CXCL8 is stimulated by
IL-1β, TNF-α, and bacterial lipopolysaccharides. In human
astrocytes, the synthesis and secretion of CXCL8 is induced by IL-1 and
TNF-α. CXCL8 is constitutively and commonly produced by various
carcinoma cell lines (Matsushima et al., 1991). In epithelial,
endothelial, and fibroblastic cells secretion of CXCL8 is induced by
IL-17 (Baggiolini et al., 1994).

[0139] CXCL8 may be of clinical relevance in psoriasis and rheumatoid
arthritis. Elevated concentrations are observed in psoriatic scales and
this may explain the high proliferation rate observed in these cells
(Gillitzer et al., 1991). CXCL8 may be also a marker of different
inflammatory processes. CXCL8 probably plays a role in the pathogenesis
of chronic polyarthritis because excessive amounts of this factor are
found in synovial fluids (Peichl et al., 1991). The activation of
neutrophils may enhance the migration of cells into the capillaries of
the joints. These cells are thought to pass through the capillaries and
enter the surrounding tissues thus causing a constant stream of
inflammatory cells through the joints.

[0140] In humans, interleukin-10 (IL-10) is produced by activated CD8(+)
peripheral blood T-cells, by T-helper CD4(+) T-cell clones (resembling
Th0, Th1, and Th2) after both antigen-specific and polyclonal activation,
by B-cell lymphomas, and by monocytes following cell activation by
bacterial lipopolysaccharides and mast cells (Howard et al., 1992).
B-cell lines derived from patients with acquired immunodeficiency
syndrome and Burkitt's lymphoma constitutively secrete large quantities
of IL-10 into the conditioned medium (de Waal-Malefyt et al., 1992). The
synthesis of IL-10 by monocytes is inhibited by IL-4 and IL-10 (Zlotnik
et al., 1991). IL-10 has been detected in the sera of a subgroup of
patients with active non-Hodgkin's lymphoma. IL-10 levels appear to
correlate with a poor survival in patients with intermediate or
high-grade non-Hodgkin's lymphoma (Blay et al., 1993).

[0141] Interleukin-12 (IL-12) is secreted by peripheral lymphocytes after
induction (Trinchieri et al., 1992). It is produced mainly by B-cells and
to a lesser extent by T-cells (Trinchieri et al., 1993). The most
powerful inducers of IL-12 are bacteria, bacterial products, and
parasites. IL-12 is produced after stimulation with phorbol esters or
calcium ionophores by human B-lymphoblastoid cells. IL-12 may be useful
in expanding an antigen-specific T-cell population. The culture of
cytotoxic T-lymphocytes with IL-12 and low-dose IL-2 leads to
proliferation only in response to an antigen co-signal (Chehimi et al.,
1993). IL-12 has been shown to augment natural killer-cell mediated
cytotoxicity in a number of conditions, including patients with hairy
cell leukemia (Bigda et al., 1993).

[0142] Interleukin-13 (IL-13) down-modulates macrophage activity, reducing
the production of pro-inflammatory cytokines and chemokines in response
to IFN-γ or bacterial lypopolysaccharides (McKenzie et al., 1993).
IL-13 enhances the production of the IL-1 receptor antagonist (IL-1ra)
(Minty et al., 1993). IL-13 also decreases the production of nitric oxide
by activated macrophages, leading to a decrease in parasiticidal
activity. IL-13 induces human monocyte differentiation, enhances survival
time in culture, and also induces B-cell differentiation and
proliferation and isotype switching (Herbert et al., 1993). It induces
IL-4 independent IgG4 and IgE synthesis in human B-cells and germ-line
IgE heavy chain gene transcription (Punnonen et al., 1993). IL-13 induces
considerable levels of IgM and IgG, but no IgA, in cultures of highly
purified surface IgD-positive or total B-cells in the presence of an
activated CD4(+) T-cell clone. IL-13 induces proliferation and
differentiation of human B-cells activated by the CD40 ligand (Cocks et
al., 1993). IL-13 synergizes with IL-2 in regulating IFN-γ
synthesis in large granular lymphocytes. IL-13 has been shown to inhibit
strongly tissue factor expression induced by bacterial
lipopolysaccharides and to reduce the pyrogenic effects of IL-1 or TNF,
thus protecting endothelial and monocyte surfaces against inflammatory
mediator induced procoagulant changes.

[0144] Interleukin-15 (IL-15) has been shown to be produced by human fetal
astrocytes and microglia in response to IL-1β, IFN-γ, and
TNF-α and is thought to play a role in T-cell mediated immune
responses in the human central nervous system. Some of the biological
activities of IL-15 resemble those of IL-2. IL-15 stimulates
proliferation of T-cells. In addition, IL-15 is also able to induce
generation of cytolytic cells and (lymphokine activated killer) LAK
cells. IL-15 appears to function as a specific maturation factor for
NK-cells. It is also been shown to function as an NK-cell survival factor
in vivo. High affinity IL-15 binding has been observed on many lymphoid
cell types, including peripheral blood monocytes, and NK-cells.

[0146] Interleukin-17 (IL-17) functions as a mediator of angiogenesis that
stimulates vascular endothelial cell migration and cord formation and
regulates production of a variety of growth factors promoting
angiogenesis (Numasaki et at, 2002). IL-17 binds to a receptor that binds
to HVS13 (referred to as viral IL-17) and to CTLA-8 (Yao et al., 1995).

[0147] Interleukin-18 (IL-18) encodes an inducer of IFN-γ production
by T-cells (Okamura et al., 1995; Micallef et al., 1996) and natural
killer cells (Tsutsui et al., 1996) that is a more potent inducer than
IL-12 (Kikkawa et al., 2001). have demonstrated that monocytes and
macrophages produce large amounts of various IL-18 species.

[0149] IL-18 is considered one of the pro-inflammatory cytokines. An
important function of IL-18 is the regulation of functionally distinct
subsets of T-helper cells required for cell mediated immune responses
(Nakanishi et al., 2001). IL-18 functions as a growth and differentiation
factor for Th1 cells.

[0150] IL-18 is a pleiotropic cytokine. IL-18 induces activated B-cells to
produce IFN-γ that inhibits IgE production (Yoshimoto et al.,
1997). IL-18 has been shown to strongly augment the production of
IFN-γ by T-cells and NK-cells (Micallef et al., 1996). The ability
of IL-18 to enhance IFN-γ production by NK cells is dependent on
the presence of IL-12 (Walker et al., 1999). IL-18 has also been found to
enhance also the production of GM-CSF (Udagawa et al., 1997) have shown
that IL-18 produced by osteoblastic stromal cells acts via GM-CSF and not
via IFN-γ to inhibit osteoclast formation.

[0152] Granulocyte-Colony Stimulating Factor (G-CSF) is secreted by
monocytes, macrophages and neutrophils after cell activation (Demetri et
al., 1991). It is produced also by stromal cells, fibroblasts, and
endothelial cells. Epithelial carcinomas, acute myeloid leukemia cells
and various tumor cell lines (bladder carcinomas, medulloblastomas), also
express this factor. The synthesis of G-CSF can be induced by bacterial
endotoxins, TNF, IL-1, and GM-CSF. Prostaglandin E2 inhibits the
synthesis of G-CSF. In epithelial, endothelial, and fibroblastic cells
secretion of G-CSF is induced by IL-17 (Moore et al., 1991).

[0153] G-CSF can be used to expand the myeloid cell lineage (Gabrilove et
al., 1992). It has been shown that the pretreatment with recombinant
human G-CSF prior to marrow harvest can improve the graft by increasing
the total number of myeloid lineage restricted progenitor cells,
resulting in stable but not accelerated myeloid engraftment of autologous
marrow (Lieschke et al., 1992). One general effect of treatment with
G-CSF appears to be a marked reduction of severe infections and episodes
of fever, which are normally observed to occur in patients with Kostmann
syndrome (Jakubowski et al., 1989). G-CSF treatment also allows dose
intensification with various antitumor drug regimes (Gianni et al.,
1992).

[0154] Granulocyte-Monocyte-Colony Stimulating Factor (GM-CSF) is secreted
together with other factors by T-cells and macrophages following cell
activation by antigens or mitogens (Moore et al., 1991). Approximately 90
percent of the secreted colony stimulating activities are due GM-CSF
(Ruef et al., 1990). The synthesis of GM-CSF by various other cell types,
for example, endothelial cells and fibroblasts, is inducible by
TNF-α, TNF-β, IL-1, IL-2, and IFN. Some cell types express
GM-CSF constitutively (Freund et al., 1992). GM-CSF can be employed for
the physiological reconstitution of hematopoiesis in all diseases
characterized either by an aberrant maturation of blood cells or by a
reduced production of leukocytes. GM-CSF can be used also to correct
chemotherapy induced cytopenias and to counteract cytopenia-related
predisposition to infections and hemorrhages (Fan et al., 1991). Several
studies have demonstrated that the use of GM-CSF enhances tolerance to
cytotoxic drug treatment and can be used to prevent dose reductions
necessitated by the side effects of cytotoxic drug treatment (Negrin et
al., 1992). GM-CSF treatment frequently permits to increase the doses of
cytotoxic drugs per course. At present, GM-CSF represents an important
advance in bone marrow transplantation and has become a standard therapy
(Armitage et al., 1992). GM-CSF enhances the reconstitution of the
hematopoietic system in patients undergoing autologous or allogenic bone
marrow transplantation and patients with delayed engraftment after bone
marrow transplantation (Schuster et al., 1992).

[0155] Interferon-α (IFN-α) forms are produced by
monocytes/macrophages, lymphoblastoid cells, fibroblasts, and a number of
different cell types following induction by viruses, nucleic acids,
glucocorticoid hormones, and low-molecular weight substances (n-butyrate,
5-bromodeoxy uridine). All known subtypes of IFN-α show the same
antiviral antiparasitic, antiproliferative activities in suitable
bioassays although they may differ in relative activities. IFN-α
inhibits the expression of a number of cytokines in hematopoietic
progenitor cells, which in turn induce a state of competence in these
cells allowing them to pass from the GO into the S-phase of the cell
cycle.

[0156] The growth of some tumor cell types in vitro is inhibited by
IFN-α which may stimulate also the synthesis of tumor-associated
cell surface antigens. In renal carcinomas IFN-α reduces the
expression of receptors for EGF. IFN-α also inhibits the growth of
fibroblasts and monocytes in vitro. IFN-α also inhibits the
proliferation of B-cell in vitro and blocks the synthesis of antibodies.
IFN-α also selectively blocks the expression of some mitochondrial
genes.

[0157] Interferon-β (IFN-β) is produced mainly by fibroblasts
and some epithelial cell types. The synthesis of IFN-beta can be induced
by common inducers of interferons, including viruses, double-stranded
RNA, and micro-organisms. It is induced also by some cytokines such as
TNF and IL1. IFN-β is involved in the regulation of unspecific
humoral immune responses and immune responses against viral infections.
IFN-β increases the expression of HLA class I antigens and blocks
the expression of HLA class II antigens stimulated by IFN-γ.
IFN-beta stimulates the activity of NK-cells and hence antibody-dependent
cytotoxicity. The activity of T suppressor cells elicited by several
stimuli is stimulated also by IFN-β. IFN-β enhances the
synthesis of the low affinity IgE receptor CD23. In activated monocytes
IFN-β induces the synthesis of neopterin. It also enhances serum
concentrations of β2-microglobulin. IFN-β selectively inhibits
the expression of some mitochondrial genes.

[0158] Interferon-γ (IFN-γ) is produced mainly by T-cells and
natural killer cells activated by antigens, mitogens, or alloantigens (De
Maeyer et al., 1992). It is produced by lymphocytes expressing the
surface antigens CD4 and CD8 (Gray et al., 1987). Like the other
interferons, IFN-γ can be used as an antiviral and antiparasitic
agent (Stuart-Harris et al., 1992). IFN-γ has been shown to be
effective in the treatment of chronic polyarthritis (Machold et al.,
1992). This treatment, which probably involves a modulation of macrophage
activities, significantly reduces joint aches and improves various
clinical parameters and allows reduction of corticosteroid doses.
IFN-γ may be of value in the treatment of opportunistic infections
in AIDS patients. It has been shown also to reduce inflammation, clinical
symptoms, and eosinophilia in severe atopic dermatitis (Hanifin et al.,
1993).

[0160] In contrast to chemotherapeutic drugs TNF-α specifically
attacks malignant cells. Extensive preclinical studies have documented a
direct cytostatic and cytotoxic effect of TNF-α against
subcutaneous human xenografts and lymph node metastases in nude
(immunodeficient) mice, as well as a variety of immunomodulatory effects
on various immune effector cells, including neutrophils, macrophages, and
T-cells (Gifford et al., 1991).

[0161] There are some indications that inhibitors of TNF-α may be of
advantage. Because TNF-α is found in the synovial fluid of patients
suffering from arthritis, these inhibitors may be helpful in ameliorating
the disease and this has been shown to be the case in animal models of
severe collagen induced arthritis (Williams et al., 1992) and in Crohn's
disease (Derkx et al., 1993). Inhibitors may ameliorate also the severe
consequences of systemic inflammatory response syndrome. TNF-α
appears to be an important autocrine modulator promoting the survival of
hairy cell leukemia cells (Lindemann et al., 1989). TNF-α has been
shown also to protect hematopoietic progenitors against irradiation and
cytotoxic agents, suggesting that it may have some potential therapeutic
applications in aplasia induced by chemotherapy or bone marrow
transplantation (Hersh et al., 1991).

[0162] Macrophage Chemotactic Protein-1 (MCP-1), now called CCL2, belongs
to the family of chemotactic cytokines known as chemokines. CCL2 is
chemotactic for monocytes but not neutrophils (Leonard et al., 1990).
Maximal induction of migration is observed at a concentration of 10 ng/ml
(Leonard et al., 1991). Point mutations have been described at two amino
acid positions, which alter the factor so that it is then also
chemotactic for neutrophils (Beall et al., 1992). Elevated levels of CCL2
are observed in macrophage-rich atherosclerotic plaques (Yla-Herttuala et
al., 1991). The factor activates the tumoricidal activity of monocytes
and macrophages in vivo. It regulates the expression of cell surface
antigens (CD11c, CD11b) and the expression of cytokines IL-1 and IL-6
(Jiang et al., 1992). CCL2 is a potent activator of human basophils,
inducing the degranulation and the release of histamines (Bischoff et
al., 1992). In basophils activated by IL-3, IL-5, or GM-CSF, CCL2
enhances the synthesis of leukotriene C4 (Bischoff et al., 1993).

[0163] IL-1, TNF-α, PDGF, TGF-β, and LIF induce the synthesis
of CCL2 in human articular chondrocytes, which may play a role in the
initiation and progression of degenerative and inflammatory arthropathies
by promoting monocyte influx and activation in synovial joints (Villiger
et al., 1992). CCL2 has been shown to exhibit biological activities other
than chemotaxis. It can induce the proliferation and activation of killer
cells known as CHAK (CC-Chemokine activated killer), which are similar to
cells activated by IL-2 (LAK cells) (Hora et al., 1992). CCL2 is also one
of the strongest histamine inducing factors.

[0164] Macrophage inflammatory protein 1-α (MIP-1α), now
called CCL3, belong to the family of chemotactic cytokines known as
chemokines. CCL3 is one of the major factors produced by macrophages
following their stimulation with bacterial endotoxins. Both proteins are
involved in the cell activation of human granulocytes (neutrophils,
eosinophils, and basophils) and appear to be involved in acute
neutrophilic inflammation. CCL3 stimulates the production of reactive
oxygen species in neutrophils and the release of lysosomal enzymes. It
also induces the synthesis of other pro-inflammatory cytokines such as
IL-1, IL-6, and TNF in fibroblasts and macrophages. CCL3 is a potent
basophil agonist, inducing a rapid change of cytosolic free calcium, the
release of histamine and sulfido-leukotrienes, and chemotaxis.

[0165] CCL3 enhances the activities of GM-CSF and promote the growth of
more mature hematopoietic progenitor cells. CCL3 also acts as an
inhibitor of the proliferation of immature hematopoietic stem cells and
has therefore been called stem cell inhibitor. CCL3 also exhibits
biological activities other than chemotaxis. It can induce the
proliferation and activation of killer cells known as
CC-chemokine-activated killer (CHAK) cells, which are similar to cells
activated by IL-2, lymphokine-activated killer (LAK) cells.

[0166] Macrophage inflammatory protein-1β (MIP-1β), now called
CCL4, is called also endogenous pyrogen. CCL4 is one of the major factors
produced by macrophages following their stimulation with bacterial
endotoxins. CCL4 is involved in the cell activation of human granulocytes
(neutrophils, eosinophils, and basophils) and appears to be involved in
acute neutrophilic inflammation. CCL4 stimulates the production of
reactive oxygen species in neutrophils and the release of lysosomal
enzymes. It also induces the synthesis of other pro-inflammatory
cytokines such as IL-1, IL-6, and TNF-α in fibroblasts and
macrophages. CCL4 antagonizes the inductive effects of CCL3. In human
monocytes the production of CCL4 can be induced by bacterial
lipopolysaccharides and IL-7 (Lord et al., 1992).

[0167] CCL4 is most effective at augmenting adhesion of CD8(+) T-cells to
the vascular cell adhesion molecule VCAM-1 and it does so by being
present on the surface of endothelial cells complexed with endothelial
proteoglycans (Cocchi et al., 1995). CCL4 also synergises with
hematopoietic growth factors (hematopoietins). CCL4 enhances the
activities of GM-CSF and promotes the growth of more mature hematopoietic
progenitor cells. CCL4 has been shown to exhibit biological activities
other than chemotaxis. It can induce the proliferation and activation of
killer cells known as CHAK (CC-Chemokine activated killer) cells, which
are similar to cells activated by IL-2 (LAK cells).

[0168] RANTES (regulated upon activation, normal T-cell expressed, and
presumably secreted) is now called CCL5 (CCL5). CCL5 belongs to the
family of chemotactic cytokines known as chemokines. CCL5 is expressed by
an early response gene. Synthesis of CCL5 is induced by TNF-α and
IL-1α.

[0169] The expression of CCL5 is inhibited following stimulation of
T-lymphocytes. CCL5 is chemotactic for T-cells, human eosinophils and
basophils and plays an active role in recruiting leukocytes into
inflammatory sites. CCL5 also activates eosinophils to release, for
example, eosinophilic cationic protein. It changes the density of
eosinophils and makes them hypodense, which is thought to represent a
state of generalized cell activation and is associated most often with
diseases such as asthma and allergic rhinitis. CCL5 also is a potent
eosinophil-specific activator of oxidative metabolism.

[0170] CCL5 increases the adherence of monocytes to endothelial cells. It
selectively supports the migration of monocytes and T-lymphocytes
expressing the cell surface markers CD4 and UCHL1. These cells are
thought to be pre-stimulated T-helper cells with memory T-cell functions.
CCL5 can induce the proliferation and activation of killer cells known as
chemokine-activated killer (CHAK) cells. CCL5 is expressed by human
synovial fibroblasts and may participate, therefore, in the ongoing
inflammatory process in rheumatoid arthritis.

[0171] Variable endothelial growth factor (VEGF) is a highly specific
mitogen for vascular endothelial cells. VEGF does not appear to enhance
the proliferation of other cell types. VEGF significantly influence
vascular permeability and is a strong angiogenic protein in several
bioassays and is thought to plays a role in neovascularisation under
physiological conditions. A potent synergism between VEGF and fibroblast
growth factor basic (bFGF) in the induction of angiogenesis has been
observed. VEGF is released from smooth muscle cells and macrophages and
is thought to play a role in the development of arteriosclerotic
diseases. In endothelial cells VEGF induces the synthesis of von
Willebrand factor. It is also a potent chemoattractant for monocytes and
thus has procoagulatory activities. In microvascular endothelial cells
VEGF induces the synthesis of plasminogen activator and plasminogen
activator inhibitor type-1. VEGF also induces the synthesis of the
metalloproteinase, interstitial collagenase, which degrades interstitial
collagen type 1, collagen type 2, and collagen type 3 under normal
physiological conditions.

[0172] VEGF is important in the pathophysiology of neuronal and other
tumors, probably functioning as a potent promoter of angiogenesis for
human gliomas. Its synthesis is induced also by hypoxia. The
extravasation of cells observed as a response to VEGF may be an important
factor determining the colonization of distant sites. Due to its
influences on vascular permeability VEGF may be involved also in altering
blood-brain-barrier functions under normal and pathological conditions.

[0173] Epidermal growth factor (EGF) has been shown to inhibit the
secretion of gastric acids. It also modulates the synthesis of a number
of hormones, including the secretion of prolactin. In the central nervous
system EGF influences the activity of some types of GABAergic and
dopaminergic neurons. EGF is a strong mitogen for many cells or
ectodermal, mesodermal, and endodermal origin. EGF controls and
stimulates the proliferation of epidermal and epithelial cells, including
fibroblasts, kidney epithelial cells, human glial cells, ovary granulosa
cells, and thyroid cells. EGF acts as a differentiation factor for some
cell types. It strongly influences the synthesis and turn-over of
proteins of the extracellular matrix, including fibronectin, collagens,
laminin, and glycosaminoglycans. EGF increases the release of calcium
from bone tissue and, like TGF-α, thus promotes bone resorption. To
a limited extent EGF also augments angiogenesis because it is mitogenic
for endothelial cells. The mitogenic activity of EGF for endothelial
cells can be potentiated by thrombin.

[0174] EGF is a strong chemoattractant for fibroblasts and epithelial
cells. EGF alone and also in combination with other cytokines is an
important factor mediating wound healing processes. EGF may be a trophic
substance for the gastrointestinal mucosa and may play a gastroprotective
role due to its ability to stimulate the proliferation of mucosa cells.
EGF has been shown to effectively promote healing of ulcers at
concentrations that do not inhibit the synthesis of gastric acids.

[0175] Fibroblast growth factors (FGF) constitutes a family of related
16-18 kDa proteins controlling normal growth and differentiation of
mesenchymal, epithelial, and neuroectodermal cell types. FGF-2 (formerly
known as basic FGF or bFGF) is the prototype of the FGF family.

[0176] Eotaxin (CCL11) is a chemokine that is a potent stimulator of
eosinophils in vitro. CCL11 does not possess suppressive activity against
immature subsets of myeloid progenitors stimulated to proliferate by
multiple growth factors (Broxmeyer et al., [INSERT]). Bartels et al.
([INSERT]), have demonstrated the presence of CCL11 sequence variants and
of low constitutive CCL11 mRNA expression in human dermal fibroblasts,
which is upregulated by IL-1α or TNF-α within 6 hrs and
modulated by IFN-γ. Induction by IL-1α is transient while
long-term stimulation with TNF-α results in a further increase of
CCL11 mRNA.

II. DISEASE STATES

[0177] A. Ankylosing Spondylitis.

[0178] AS is a disease subset within a broader disease classification of
spondyloarthropathy. Patients affected with the various subsets of
spondyloarthropathy have disease etiologies that are often very
different, ranging from bacterial infections to inheritance. Yet, in all
subgroups, the end result of the disease process is axial arthritis.
Despite the early clinically differences seen in the various patient
populations, many of them end up nearly identical after a disease course
of ten-to-twenty years. Recent studies suggest the mean time to clinical
diagnosis of ankylosing spondylitis from disease onset of disease is 7.5
years (Khan, 1998). These same studies suggest that the
spondyloarthropathies may have prevalence close to that of rheumatoid
arthritis (Feldtkeller et al., 2003; Doran et al., 2003).

[0179] AS is a chronic systemic inflammatory rheumatic disorder of the
axial skeleton with or without extraskeletal manifestations. Sacroiliac
joints and the spine are primarily affected, but hip and shoulder joints,
and less commonly peripheral joints or certain extra-articular structures
such as the eye, vasculature, nervous system, and gastrointestinal system
may also be involved. Its etiology is not yet fully understood
(Wordsworth, 1995; Calin and Taurog, 1998). It is strongly associated
with the major histocompatibility class I (MHC I) HLA-B27 allele (Calin
and Taurog, 1998). AS affects individuals in the prime of their life and
is feared because of its potential to cause chronic pain and irreversible
damage of tendons, ligaments, joints, and bones (Brewerton et al., 1973;
Brewerton et al., 1973; Schlosstein et al., 1973). AS may occur alone or
in association with another form of spondyloarthropathy such as reactive
arthritis, psoriasis, psoriatic arthritis, enthesitis, ulcerative
colitis, irritable bowel disease, or Crohn's disease, in which case it is
classified as secondary AS.

[0180] Typically, the affected sites include the discovertebral,
apophyseal, costovertebral, and costotransverse joints of the spine, and
the paravertebral ligamentous structures. Inflammation of the entheses,
which are sites of musculotendinous and ligamentous attachment to bones,
is also prominent in this disease (Calin and Taurog, 1998). The site of
enthesitis is known to be infiltrated by plasma cells, lymphocytes, and
polymorphonuclear cells. The inflammatory process frequently results in
gradual fibrous and bony ankylosis, (Ball, 1971; Khan, 1990).

[0181] Delayed diagnosis is common because symptoms are often attributed
to more common back problems. A dramatic loss of flexibility in the
lumbar spine is an early sign of AS. Other common symptoms include
chronic pain and stiffness in the lower back which usually starts where
the lower spine is joined to the pelvis, or hip.

[0182] Although most symptoms begin in the lumbar and sacroiliac areas,
they may involve the neck and upper back as well. Arthritis may also
occur in the shoulder, hips and feet. Some patients have eye
inflammation, and more severe cases must be observed for heart valve
involvement.

[0183] The most frequent presentation is back pain, but disease can begin
atypically in peripheral joints, especially in children and women, and
rarely with acute iritis (anterior uveitis). Additional early symptoms
and signs are diminished chest expansion from diffuse costovertebral
involvement, low-grade fever, fatigue, anorexia, weight loss, and anemia.
Recurrent back pain--often nocturnal and of varying intensity--is an
eventual complaint, as is morning stiffness typically relieved by
activity. A flexed or bent-over posture eases back pain and paraspinal
muscle spasm; thus, some degree of kyphosis is common in untreated
patients.

[0184] Systemic manifestations occur in 1/3 of patients. Recurrent,
usually self-limited, acute iritis (anterior uveitis) rarely is
protracted and severe enough to impair vision. Neurologic signs can
occasionally result from compression radiculitis or sciatica, vertebral
fracture or subluxation, and cauda equina syndrome (which consists of
impotence, nocturnal urinary incontinence, diminished bladder and rectal
sensation, and absence of ankle jerks). Cardiovascular manifestations can
include aortic insufficiency, angina, pericarditis, and ECG conduction
abnormalities. A rare pulmonary finding is upper lobe fibrosis,
occasionally with cavitation that may be mistaken for TB and can be
complicated by infection with Aspergillus.

[0185] AS is characterized by mild or moderate flares of active
spondylitis alternating with periods of almost or totally inactive
inflammation. Proper treatment in most patients results in minimal or no
disability and in full, productive lives despite back stiffness.
Occasionally, the course is severe and progressive, resulting in
pronounced incapacitating deformities. The prognosis is bleak for
patients with refractory iritis and for the rare patient with secondary
amyloidosis.

[0186] The ESR and other acute-phase reactants (e.g., C-reactive protein
and serum Ig levels) are mildly elevated in most patients with active AS.
Tests for IgM rheumatoid factor and antinuclear antibodies are negative.
A positive test for HLA-B27 is usual but not invariable and not specific
(a negative test is more useful in helping to exclude AS than a positive
test is in diagnosing it). This test is not necessary in patients with
typical disease.

[0187] Diagnosis must be confirmed by x-ray. The earliest abnormalities
(pseudo-widening from subchondral erosions, sclerosis or later narrowing)
occur in the sacroiliac joints. Early changes in the spine are upper
lumbar vertebral squaring and demineralization, spotty ligamentous
calcification, and one or two evolving syndesmophytes. The classic bamboo
spine with prominent syndesmophytes and diffuse paraspinal ligamentous
calcification is not useful for early diagnosis; these changes develop in
a minority of patients over an average period of 10 years.

[0188] The severity of joint involvement and the degree of systemic
symptoms vary greatly from one individual to another. Early, accurate
diagnosis and therapy may minimize years of pain and disability.

[0189] Joint discomfort may be relieved with drugs. Treatment plans
usually address prevention, delay, or correction of the deformity and
psychosocial and rehabilitation needs. For proper posture and joint
motion, daily exercise and other supportive measures (e.g., postural
training, therapeutic exercise) are vital to strengthen muscle groups
that oppose the direction of potential deformities (i.e., strengthen the
extensor rather than flexor muscle groups). Reading while lying prone and
thus extending the neck may help keep the back flexible.

[0190] NSAIDs facilitate exercise and other supportive measures by
suppressing articular inflammation, pain, and muscle spasm. Most NSAIDs
are of proven value in AS, but tolerance and toxicity, rather than
marginal differences in efficacy, dictate drug choice. Patients should be
monitored and warned of potential adverse reactions. The daily dose of
NSAIDs should be as low as possible, but maximum doses of a drug such as
indomethacin may be needed with active disease. Drug withdrawal should be
attempted only slowly, after systemic and articular signs of active
disease have been suppressed for several months. Several new NSAIDs,
referred to as COX-2 drugs because they inhibit cyclooxygenase-2, provide
equal effectiveness to drugs that inhibit COX-1 with less chance of
adverse effects on the gastric mucosa, and platelet aggregation.

[0191] Corticosteroids have limited therapeutic value; long-term use is
associated with many serious adverse effects, including osteoporosis of
the stiff spine. For acute iritis, topical corticosteroids (and
mydriatics) usually are adequate; oral corticosteroids are rarely
indicated. Intra-articular corticosteroids may be beneficial,
particularly when one or two peripheral joints are more severely inflamed
than others, thereby compromising exercise and rehabilitation.

[0192] Most slow-acting (remitting) drugs for RA (e.g., gold given IM)
either have not been studied or are not effective for AS. Sulfasalazine
may be helpful, particularly when the peripheral joints are involved.
Dosage should be started at 500 mg/day and increased by 500 mg/day at
1-wk intervals to 1 g bid maintenance (see also Rheumatoid Arthritis in
Ch. 50). The most common side effect is nausea, which is mainly central,
but enteric-coated tablets are better tolerated. Dose reduction may help.

[0193] Narcotics, other strong analgesics, and muscle relaxants lack
anti-inflammatory properties and should be prescribed only short-term as
adjuncts to help control severe back pain and spasm. Radiotherapy to the
spine, although effective, is recommended as a last resort because it
increases the risk of acute myelogenous leukemia ten-fold.

[0194] Rehabilitation therapies are essential. Proper sleep and walking
positions, coupled with abdominal and back exercises, help maintain
posture. Exercises help maintain joint flexibility. Breathing exercises
enhance lung capacity, and swimming provides aerobic exercise. Even with
optimal treatment, some people will develop a stiff or "ankylosed" spine,
but they will remain functional if this fusion occurs in an upright
position. Continuing care is critical. AS is a lifelong problem, and
people often fail to continue treatment, in which case permanent posture
and mobility losses occur.

[0195] B. Psoratic Arthritis

[0196] Psoriasis is an inflammatory and proliferative skin disorder with a
prevalence of 1.5-3%. Approximately 20% of patients with psoriasis
develop a characteristic form of arthritis that has several patterns
(Gladman, 1992; Moll & Wright, 1991; Jones et al., 1994; Gladman et al.,
1995). Some individuals present with joint symptoms first but in the
majority, skin psoriasis presents first. About one-third of patients have
simultaneous exacerbations of their skin and joint disease (Gladman et
al., 1987) and there is a topographic relationship between nail and
distal interphalangeal joint disease (Jones et al., 1994; 33:834-9; V.
Wright, 1956). Although the inflammatory processes which link skin, nail
and joint disease remain elusive, an immune-mediated pathology is
implicated.

[0197] Psoriatic arthritis (PsA) is a chronic inflammatory arthropathy
characterized by the association of arthritis and psoriasis and was
recognized as a clinical entity distinct from rheumatoid arthritis (RA)
in 1964 (Blumberg et al., 1964). Subsequent studies have revealed that
PsA shares a number of genetic, pathogenic and clinical features with
other spondyloarthropathies (SpAs), a group of diseases that comprise
ankylosing spondylitis, reactive arthritis and enteropathic arthritis
(Wright, V., 1979). The notion that PsA belongs to the SpA group has
recently gained further support from imaging studies demonstrating
widespread enthesitis in the, including PsA but not RA (McGonagle et al.,
1999; McGonagle et al., 1998). More specifically, enthesitis has been
postulated to be one of the earliest events occurring in the SpAs,
leading to bone remodeling and ankylosis in the spine, as well as to
articular synovitis when the inflamed entheses are close to peripheral
joints. However, the link between enthesitis and the clinical
manifestations in PsA remains largely unclear, as PsA can present with
fairly heterogeneous patterns of joint involvement with variable degrees
of severity (Marsal et al., 1999; Salvarani et al., 1998). Thus, other
factors must be posited to account for the multifarious features of PsA,
only a few of which (such as the expression of the HLA-B27 molecule,
which is strongly associated with axial disease) have been identified. As
a consequence, it remains difficult to map the disease manifestations to
specific pathogenic mechanisms, which means that the treatment of this
condition remains largely empirical.

[0198] Family studies have suggested a genetic contribution to the
development of PsA (Moll & Wright, 1973). Other chronic inflammatory
forms of arthritis, such as ankylosing spondylitis and rheumatoid
arthritis, are thought to have a complex genetic basis. However, the
genetic component of PsA has been difficult to assess for several
reasons. There is strong evidence for a genetic predisposition to
psoriasis alone that may mask the genetic factors that are important for
the development of PsA. Although most would accept PsA as a distinct
disease entity, at times there is a phenotypic overlap with rheumatoid
arthritis and ankylosing spondylitis. Also, PsA itself is not a
homogeneous condition and various subgroups have been proposed. Although
not all these confounding factors were overcome in the present study, we
concentrated on investigating candidate genes in three broad categories
of patients with PsA that cover the disease spectrum.

[0199] Polymorphisms in the promoter region of the TNFA region are of
considerable interest as they may influence levels of TNF-α
secretion (Jacob et al., 1990; Bendzen et al., 1988). Increased amounts
of TNF-α have been reported in both psoriatic skin (Ettehadi et
al., 1994) and synovial fluid (Partsch et al., 1997).

[0200] Recent trials have shown a positive benefit of anti-TNF treatment
in both PsA (Mease et al., 2000) and ankylosing spondylitis (Brandt et
al., 2000). Furthermore, the locus for TNF-α resides within the
class DI region of the MHC and thus may provide tighter associations with
PsA than those provided by flanking class I and class II regions. There
were relatively weak associations with the TNFA alleles in our total PsA
group. The uncommon TNFA-238A allele was increased in frequency in the
group with peripheral polyarthritis and absent in those patients with
spondylitis, although this finding may be explained by linkage
disequilibrium with HLA-Cw*0602. Whether there are functional
consequences associated with polymorphisms at the TNFA-238 allele is
unclear (Pociot et al., 1995). Nonetheless, it is possible that the
pattern of arthritis that develops in patients with psoriasis may be
linked directly or indirectly to this particular allele.

[0201] Hohler et al. (A TNF-α promoter polymorphism is associated
with juvenile onset psoriasis and psoriatic arthritis, J Invest Dermatol,
1997; 109:562-5) found an increase in the frequency of the TNFA-238A
allele in patients with PsA as well as in juvenile onset psoriasis. The
association of TNFA-238A with both juvenile onset psoriasis and PsA was
stronger than that with HLA-Cw6. Similarly, in our study, there were
strong associations between juvenile onset psoriasis and both HLA-Cw*0602
and TNFA-238A, although neither allele had any relationship to the age of
onset of arthritis. In our study, all patients with PsA who had at least
one TNFA-238A allele were HLA-Cw6-positive, emphasizing the close linkage
between these alleles in PsA. However, in contrast to the study by Hohler
et al. (1997), and explainable by close linkage to HLA-Cw*0602, the
TNFA-238A allele was only increased in patients with peripheral
arthritis. It is also of interest that, in a separate study of ankylosing
spondylitis, the same group found the uncommon TNFA-308A and -238A
alleles to have a protective effect on the development of spondylitis
(Hohler et al., Association of different tumor necrosis factor alpha
promoter allele frequencies with ankylosing spondylitis in HLA-B27
positive individuals. Arthritis Rheum 1998; 41:1489-92).

[0204] It has been argued that clearance of reactive arthritis-associated
bacteria requires the production of appropriate levels of IFN-γ and
TNF-α, while IL-10 acts by suppressing these responses (Autenrieth
et al., 1994; Sieper & Braun, 1995). IL-10 is a regulatory cytokine that
inhibits the synthesis of IL-12 and TNF-γ by activated macrophages
(de Waal et al., 1991; Hart et al., 1995; Chomarat et al., 1995) and of
IFN-γ by T cells (Macatonia et al., 1993).

[0205] D. Enteropathic Arthritis

[0206] Enteropathic arthritis (EA) occurs in combination with inflammatory
bowel diseases (IBD) such as Crohn's disease or ulcerative colitis. It
also can affect the spine and sacroiliac joints. Enteropathic arthritis
involves the peripheral joints, usually in the lower extremities such as
the knees or ankles. It commonly involves only a few or a limited number
of joints and may closely follow the bowel condition. This occurs in
approximately 11% of patients with ulcerative colitis and 21% of those
with Crohn's disease. The synovitis is generally self-limited and
non-deforming.

[0208] The precise causes of enteropathic arthropathies are unknown.
Inflammation of the GI tract may increase permeability, resulting in
absorption of antigenic material, including bacterial antigens. These
arthrogenic antigens may then localize in musculoskeletal tissues
(including entheses and synovium), thus eliciting an inflammatory
response. Alternatively, an autoimmune response may be induced through
molecular mimicry, in which the host's immune response to these antigens
cross-reacts with self-antigens in synovium.

[0209] Of particular interest is the strong association between reactive
arthritis and HLA-B27, an HLA class 1 molecule. A potentially
arthrogenic, bacterially derived antigen peptide could fit in the
antigen-presenting groove of the B27 molecule, resulting in a CD8+ T-cell
response. HLA-B27 transgenic rats develop features of enteropathic
arthropathy with arthritis and gut inflammation.

[0210] E. Ulcerative Colitis

[0211] Ulcerative colitis is a disease that causes inflammation and sores,
called ulcers, in the lining of the large intestine. The inflammation
usually occurs in the rectum and lower part of the colon, but it may
affect the entire colon. Ulcerative colitis rarely affects the small
intestine except for the end section, called the terminal ileum.
Ulcerative colitis may also be called colitis or proctitis. The
inflammation makes the colon empty frequently, causing diarrhea. Ulcers
form in places where the inflammation has killed the cells lining the
colon; the ulcers bleed and produce pus.

[0212] Ulcerative colitis is an inflammatory bowel disease (IBD), the
general name for diseases that cause inflammation in the small intestine
and colon. Ulcerative colitis can be difficult to diagnose because its
symptoms are similar to other intestinal disorders and to another type of
IBD, Crohn's disease. Crohn's disease differs from ulcerative colitis
because it causes inflammation deeper within the intestinal wall. Also,
Crohn's disease usually occurs in the small intestine, although it can
also occur in the mouth, esophagus, stomach, duodenum, large intestine,
appendix, and anus.

[0213] Ulcerative colitis may occur in people of any age, but most often
it starts between ages 15 and 30, or less frequently between ages 50 and
70. Children and adolescents sometimes develop the disease. Ulcerative
colitis affects men and women equally and appears to run in some
families. Theories about what causes ulcerative colitis abound, but none
have been proven. The most popular theory is that the body's immune
system reacts to a virus or a bacterium by causing ongoing inflammation
in the intestinal wall. People with ulcerative colitis have abnormalities
of the immune system, but doctors do not know whether these abnormalities
are a cause or a result of the disease. Ulcerative colitis is not caused
by emotional distress or sensitivity to certain foods or food products,
but these factors may trigger symptoms in some people.

[0214] The most common symptoms of ulcerative colitis are abdominal pain
and bloody diarrhea. Patients also may experience fatigue, weight loss,
loss of appetite, rectal bleeding, and loss of body fluids and nutrients.
About half of patients have mild symptoms. Others suffer frequent fever,
bloody diarrhea, nausea, and severe abdominal cramps. Ulcerative colitis
may also cause problems such as arthritis, inflammation of the eye, liver
disease (hepatitis, cirrhosis, and primary sclerosing cholangitis),
osteoporosis, skin rashes, and anemia. No one knows for sure why problems
occur outside the colon. Scientists think these complications may occur
when the immune system triggers inflammation in other parts of the body.
Some of these problems go away when the colitis is treated.

[0215] A thorough physical exam and a series of tests may be required to
diagnose ulcerative colitis. Blood tests may be done to check for anemia,
which could indicate bleeding in the colon or rectum. Blood tests may
also uncover a high white blood cell count, which is a sign of
inflammation somewhere in the body. By testing a stool sample, the doctor
can detect bleeding or infection in the colon or rectum. The doctor may
do a colonoscopy or sigmoidoscopy. For either test, the doctor inserts an
endoscope--a long, flexible, lighted tube connected to a computer and TV
monitor--into the anus to see the inside of the colon and rectum. The
doctor will be able to see any inflammation, bleeding, or ulcers on the
colon wall. During the exam, the doctor may do a biopsy, which involves
taking a sample of tissue from the lining of the colon to view with a
microscope. A barium enema x ray of the colon may also be required. This
procedure involves filling the colon with barium, a chalky white
solution. The barium shows up white on x-ray film, allowing the doctor a
clear view of the colon, including any ulcers or other abnormalities that
might be there.

[0216] Treatment for ulcerative colitis depends on the seriousness of the
disease. Most people are treated with medication. In severe cases, a
patient may need surgery to remove the diseased colon. Surgery is the
only cure for ulcerative colitis. Some people whose symptoms are
triggered by certain foods are able to control the symptoms by avoiding
foods that upset their intestines, like highly seasoned foods, raw fruits
and vegetables, or milk sugar (lactose). Each person may experience
ulcerative colitis differently, so treatment is adjusted for each
individual. Emotional and psychological support is important. Some people
have remissions--periods when the symptoms go away--that last for months
or even years. However, most patients' symptoms eventually return. This
changing pattern of the disease means one cannot always tell when a
treatment has helped. Some people with ulcerative colitis may need
medical care for some time, with regular doctor visits to monitor the
condition.

[0217] The goal of therapy is to induce and maintain remission, and to
improve the quality of life for people with ulcerative colitis. Several
types of drugs are available: [0218] Aminosalicylates--drugs that
contain 5-aminosalicyclic acid (5-ASA), help control inflammation.
Sulfasalazine is a combination of sulfapyridine and 5-ASA and is used to
induce and maintain remission. The sulfapyridine component carries the
anti-inflammatory 5-ASA to the intestine. However, sulfapyridine may lead
to side effects such as include nausea, vomiting, heartburn, diarrhea,
and headache. Other 5-ASA agents such as olsalazine, mesalamine, and
balsalazide, have a different carrier, offer fewer side effects, and may
be used by people who cannot take sulfasalazine. 5-ASAs are given orally,
through an enema, or in a suppository, depending on the location of the
inflammation in the colon. Most people with mild or moderate ulcerative
colitis are treated with this group of drugs first. [0219]
Corticosteroids--such as prednisone and hydrocortisone also reduce
inflammation. They may be used by people who have moderate to severe
ulcerative colitis or who do not respond to 5-ASA drugs. Corticosteroids,
also known as steroids, can be given orally, intravenously, through an
enema, or in a suppository, depending on the location of the
inflammation. These drugs can cause side effects such as weight gain,
acne, facial hair, hypertension, mood swings, and an increased risk of
infection. For this reason, they are not recommended for long-term use.
[0220] Immunomodulators--such as azathioprine and 6-mercapto-purine
(6-MP) reduce inflammation by affecting the immune system. They are used
for patients who have not responded to 5-ASAs or corticosteroids or who
are dependent on corticosteroids. However, immunomodulators are
slow-acting and may take up to 6 months before the full benefit is seen.
Patients taking these drugs are monitored for complications including
pancreatitis and hepatitis, a reduced white blood cell count, and an
increased risk of infection. Cyclosporine A may be used with 6-MP or
azathioprine to treat active, severe ulcerative colitis in people who do
not respond to intravenous corticosteroids. Other drugs may be given to
relax the patient or to relieve pain, diarrhea, or infection.

[0221] Occasionally, symptoms are severe enough that the person must be
hospitalized. For example, a person may have severe bleeding or severe
diarrhea that causes dehydration. In such cases the doctor will try to
stop diarrhea and loss of blood, fluids, and mineral salts. The patient
may need a special diet, feeding through a vein, medications, or
sometimes surgery.

[0222] About 25-40% of ulcerative colitis patients must eventually have
their colons removed because of massive bleeding, severe illness, rupture
of the colon, or risk of cancer. Sometimes the doctor will recommend
removing the colon if medical treatment fails or if the side effects of
corticosteroids or other drugs threaten the patient's health. Surgery to
remove the colon and rectum, known as proctocolectomy, is followed by one
of the following: [0223] Ileostomy, in which the surgeon creates a
small opening in the abdomen, called a stoma, and attaches the end of the
small intestine, called the ileum, to it. Waste will travel through the
small intestine and exit the body through the stoma. The stoma is about
the size of a quarter and is usually located in the lower right part of
the abdomen near the beltline. A pouch is worn over the opening to
collect waste, and the patient empties the pouch as needed. [0224]
Ileoanal anastomosis, or pull-through operation, which allows the patient
to have normal bowel movements because it preserves part of the anus. In
this operation, the surgeon removes the diseased part of the colon and
the inside of the rectum, leaving the outer muscles of the rectum. The
surgeon then attaches the ileum to the inside of the rectum and the anus,
creating a pouch. Waste is stored in the pouch and passed through the
anus in the usual manner. Bowel movements may be more frequent and watery
than before the procedure. Inflammation of the pouch (pouchitis) is a
possible complication. Not every operation is appropriate for every
person. Which surgery to have depends on the severity of the disease and
the patient's needs, expectations, and lifestyle. People faced with this
decision should get as much information as possible by talking to their
doctors, to nurses who work with colon surgery patients (enterostomal
therapists), and to other colon surgery patients. Patient advocacy
organizations can direct people to support groups and other information
resources.

[0225] Most people with ulcerative colitis will never need to have
surgery. If surgery does become necessary, however, some people find
comfort in knowing that after the surgery, the colitis is cured and most
people go on to live normal, active lives.

[0226] F. Crohn's Disease

[0227] Another disorder for which immunosuppression has been tried is
Crohn's disease. Crohn's disease symptoms include intestinal inflammation
and the development of intestinal stenosis and fistulas; neuropathy often
accompanies these symptoms. Anti-inflammatory drugs, such as
5-aminosalicylates (e.g., mesalamine) or corticosteroids, are typically
prescribed, but are not always effective (reviewed in V. A. Botoman et
al., 1998). Immunosuppression with cyclosporine is sometimes beneficial
for patients resistant to or intolerant of corticosteroids (J. Brynskov
et al., 1989).

[0229] One hypothesis for the etiology of Crohn's disease is that a
failure of the intestinal mucosal barrier, possibly resulting from
genetic susceptibilities and environmental factors (e.g., smoking),
exposes the immune system to antigens from the intestinal lumen including
bacterial and food antigens (e.g., Soderholm et al., 1999; Hollander et
al., 1986; D. Hollander, 1992). Another hypothesis is that persistent
intestinal infection by pathogens such as Mycobacterium paratuberculosis,
Listeria monocytogenes, abnormal Escherichia coli, or paramyxovirus,
stimulates the immune response; or alternatively, symptoms result from a
dysregulated immune response to ubiquitous antigens, such as normal
intestinal microflora and the metabolites and toxins they produce
(Sartor, 1997). The presence of IgA and IgG anti-Sacccharomyces
cerevisiae antibodies (ASCA) in the serum was found to be highly
diagnostic of pediatric Crohn's disease (Ruemmele et al., 1998;
Hoffenberg et al., 1999).

[0231] Recent efforts to develop diagnostic and treatment tools against
Crohn's disease have focused on the central role of cytokines (Schreiber,
1998; van Hogezand & Verspaget, 1998). Cytokines are small secreted
proteins or factors (5 to 20 kD) that have specific effects on
cell-to-cell interactions, intercellular communication, or the behavior
of other cells. Cytokines are produced by lymphocytes, especially
TH1 and TH2 lymphocytes, monocytes, intestinal macrophages,
granulocytes, epithelial cells, and fibroblasts (reviewed in Rogler &.
Andus, 1998; Galley & Webster, 1996). Some cytokines are pro-inflammatory
(e.g., TNF-α, IL-1(α and β), IL-6, IL-8, IL-12, or
leukemia inhibitory factor [LIF]); others are anti-inflammatory (e.g.,
IL-1 receptor antagonist, IL-4, IL-10, IL-11, and TGF-β). However,
there may be overlap and functional redundancy in their effects under
certain inflammatory conditions.

[0232] In active cases of Crohn's disease, elevated concentrations of
TNF-α and IL-6 are secreted into the blood circulation, and
TNF-α, IL-1, IL-6, and IL-8 are produced in excess locally by
mucosal cells (id.; Funakoshi et al., 1998). These cytokines can have
far-ranging effects on physiological systems including bone development,
hematopoiesis, and liver, thyroid, and neuropsychiatric function. Also,
an imbalance of the IL-1β/IL-1ra ratio, in favor of pro-inflammatory
IL-1β, has been observed in patients with Crohn's disease (Rogler &
Andus, 1998; Saiki et al., 1998; Dionne et al., 1998; but see S.
Kuboyama, 1998). One study suggested that cytokine profiles in stool
samples could be a useful diagnostic tool for Crohn's disease (Saiki et
al., 1998).

[0234] Another approach to the treatment of Crohn's disease has focused on
at least partially eradicating the bacterial community that may be
triggering the inflammatory response and replacing it with a
non-pathogenic community. For example, U.S. Pat. No. 5,599,795 discloses
a method for the prevention and treatment of Crohn's disease in human
patients. Their method was directed to sterilizing the intestinal tract
with at least one antibiotic and at least one anti-fungal agent to kill
off the existing flora and replacing them with different, select,
well-characterized bacteria taken from normal humans. Borody taught a
method of treating Crohn's disease by at least partial removal of the
existing intestinal microflora by lavage and replacement with a new
bacterial community introduced by fecal inoculum from a disease-screened
human donor or by a composition comprising Bacteroides and Escherichia
coli species. (U.S. Pat. No. 5,443,826). However, there has been no known
cause of Crohn's disease to which diagnosis and/or treatment could be
directed.

[0235] G. Rhematoid Arthritis

[0236] The exact etiology of RA remains unknown, but the first signs of
joint disease appear in the synovial lining layer, with proliferation of
synovial fibroblasts and their attachment to the articular surface at the
joint margin (Lipsky, 1998). Subsequently, macrophages, T cells and other
inflammatory cells are recruited into the joint, where they produce a
number of mediators, including the cytokines interleukin-1 (IL-1), which
contributes to the chronic sequelae leading to bone and cartilage
destruction, and tumour necrosis factor (TNF-α), which plays a role
in inflammation (Dinarello, 1998; Arend & Dayer, 1995; van den Berg,
2001). The concentration of IL-1 in plasma is significantly higher in
patients with RA than in healthy individuals and, notably, plasma IL-1
levels correlate with RA disease activity (Eastgate et al., 1988).
Moreover, synovial fluid levels of IL-1 are correlated with various
radiographic and histologic features of RA (Kahle et al., 1992; Rooney et
al., 1990).

[0237] In normal joints, the effects of these and other proinflammatory
cytokines are balanced by a variety of anti-inflammatory cytokines and
regulatory factors (Burger & Dayer, 1995). The significance of this
cytokine balance is illustrated in juvenile RA patients, who have
cyclical increases in fever throughout the day (Prieur et al., 1987).
After each peak in fever, a factor that blocks the effects of IL-1 is
found in serum and urine. This factor has been isolated, cloned and
identified as IL-1 receptor antagonist (IL-1ra), a member of the IL-1
gene family (Hannum et al., 1990). IL-1ra, as its name indicates, is a
natural receptor antagonist that competes with IL-1 for binding to type I
IL-1 receptors and, as a result, blocks the effects of IL-1 (Arend et
al., 1998). A 10- to 100-fold excess of IL-1ra may be needed to block
IL-1 effectively; however, synovial cells isolated from patients with RA
do not appear to produce enough IL-1ra to counteract the effects of IL-1
(Firestein et al., 1994; Fujikawa et al., 1995).

[0238] H. Systemic Lupus Erythematosus

[0239] There has also been no known cause for autoimmune diseases such as
systemic lupus erythematosus. Systemic lupus erythematosus (SLE) is an
autoimmune rheumatic disease characterized by deposition in tissues of
autoantibodies and immune complexes leading to tissue injury (Kotzin,
1996). In contrast to autoimmune diseases such as MS and type 1 diabetes
mellitus, SLE potentially involves multiple organ systems directly, and
its clinical manifestations are diverse and variable (reviewed by Kotzin
& O'Dell, 1995). For example, some patients may demonstrate primarily
skin rash and joint pain, show spontaneous remissions, and require little
medication. At the other end of the spectrum are patients who demonstrate
severe and progressive kidney involvement that requires therapy with high
doses of steroids and cytotoxic drugs such as cyclophosphamide (Kotzin,
1996).

[0240] The serological hallmark of SLE, and the primary diagnostic test
available, is elevated serum levels of IgG antibodies to constituents of
the cell nucleus, such as double-stranded DNA (dsDNA), single-stranded
DNA (ss-DNA), and chromatin. Among these autoantibodies, IgG anti-dsDNA
antibodies play a major role in the development of lupus
glomerulonephritis (G N) (Hahn & Tsao, 1993; Ohnishi et al., 1994).
Glomerulonephritis is a serious condition in which the capillary walls of
the kidneys blood purifying glomeruli become thickened by accretions on
the epithelial side of glomerular basement membranes. The disease is
often chronic and progressive and may lead to eventual renal failure.

[0241] The mechanisms by which autoantibodies are induced in these
autoimmune diseases remains unclear. As there has been no known cause of
SLE, to which diagnosis and/or treatment could be directed, treatment has
been directed to suppressing immune responses, for example with macrolide
antibiotics, rather than to an underlying cause. (e.g., U.S. Pat. No.
4,843,092).

[0242] I. Familial Mediterranean Fever

[0243] Familial Mediterranean Fever is an inherited disorder usually
characterized by recurrent episodes of fever and peritonitis
(inflammation of the abdominal membrane). In 1997, researchers identified
the gene for FMF and found several different gene mutations that cause
this inherited rheumatic disease. The gene, found on chromosome 16, codes
for a protein that is found almost exclusively in granulocytes--white
blood cells important in the immune response. The protein is likely to
normally assist in keeping inflammation under control by deactivating the
immune response--without this `brake,` an inappropriate full-blown
inflammatory reaction occurs: an attack of FMF. To explore whether a
molecular diagnostic cytokine characteristic exists, serum samples from
six patients with clinically diagnosed FMF were examined and the
concentration of cytokines were quantified.

[0244] J. Amyotrophic Lateral Sclerosis

[0245] Amyotrophic lateral sclerosis (ALS) is an invariably fatal, usually
rapidly-progressing disease that kills motor neurons in the brainstem,
spinal cord and motor cortex. Approximately 85% of all ALS cases are
sporadic. Of the remaining familial fraction, approximately 25% are
caused by mutations in Cu,Zn-superoxide dismutase (SOD1) (Cudkowicz et
al., 1997; Orrell et al., 2000). A murine model for familial ALS has been
in existence for ten years (Gurney et al., 1994). In particular mice
expressing high copy numbers of human mutant SOD1 with glycine to alanine
substituted at residue 93 (G93A-SOD1) develop motor neuron disease at 3-4
months of age. Disease onset in these mice is marked, in part, by
broad-spectrum upregulation of pro-inflammatory cytokines and selective
up-regulation of some archetypal anti-inflammatory cytokines within the
central nervous system (Hensley et al., 2002; 2003). Other research has
implicated arachidonate metabolites, particularly prostaglandins produced
by cyclooxygeanse-II (COX-II), in the pathobiology of murine ALS
(Drachman et al., 2002). The observation of progressive neuroinflammation
in the G93A-SOD1 mouse, combined with recent reports that neuron-specific
expression of mutant SOD1 fails to recapitulate disease (Pramatarova et
al., 2001; Lino et al., 2002), has lent credence to the hypothesis that
glial dysfunction precipitates neuropathology in the murine model of ALS
perhaps through disruption of normal cytokine homoeostasis.

[0246] To date, few studies have been performed to survey cytokine
alterations in humans with ALS. Cytokine networks are technically
difficult to study because of the dynamic inter-relationships that exist
amongst antagonizing or synergistic cytokines. Additionally, human ALS
studies may be complicated in the latter stages by general tissue
degradation and confounding effects from pharmacotherapy. This is very
unfortunate, because there is an urgent need for biomarkers of disease
severity (and of drug efficacy) that can be applied to human clinical
studies (Bradley et al., 2003). Development of robust biomarkers would
facilitate the clinical evaluation of therapeutic candidates, by allowing
the assessment of drug efficacy sooner than would be possible through
traditional instruments such as function-of-living questionnaires and
survival analyses.

[0247] K. Irritable Bowel Syndrome

[0248] Irritable bowel syndrome (IBS) is a functional disorder
characterized by abdominal pain and altered bowel habits. This syndrome
may begin in young adulthood and can be associated with significant
disability. This syndrome is not a homogeneous disorder. Rather, subtypes
of IBS have been described on the basis of the predominant
symptom--diarrhea, constipation, or pain. In the absence of "alarm"
symptoms, such as fever, weight loss, and gastrointestinal bleeding, a
limited workup is needed. Once a diagnosis of IBS is made, an integrated
treatment approach can effectively reduce the severity of symptoms. IBS
is a common disorder, although its prevalence rates have varied. In
general, IBS affects about 15% of US adults and occurs about three times
more often in women than in men (Jailwala et al., 2000).

[0249] IBS accounts for between 2.4 million and 3.5 million visits to
physicians each year. It not only is the most common condition seen by
gastroenterologists but also is one of the most common gastrointestinal
conditions seen by primary care physicians (Everhart et al., 1991;
Sandler, 1990).

[0250] IBS is also a costly disorder. Compared with persons who do not
have bowel symptoms, persons with IBS miss three times as many workdays
and are more likely to report being too sick to work (Drossman et al.,
1993; Drossman et al., 1997). Moreover, those with IBS incur hundreds of
dollars more in medical charges than persons without bowel disorders
(Talley et al., 1995).

[0251] No specific abnormality accounts for the exacerbations and
remissions of abdominal pain and altered bowel habits experienced by
patients with IBS. The evolving theory of IBS suggests dysregulation at
multiple levels of the brain-gut axis. Dysmotility, visceral
hypersensitivity, abnormal modulation of the central nervous system
(CNS), and infection have all been implicated. In addition, psychosocial
factors play an important modifying role. Abnormal intestinal motility
has long been considered a factor in the pathogenesis of IBS. Transit
time through the small intestine after a meal has been shown to be
shorter in patients with diarrhea-predominant IBS than in patients who
have the constipation-predominant or pain-predominant subtype (Cann et
al., 1983).

[0252] In studies of the small intestine during fasting, the presence of
both discrete, clustered contractions and prolonged, propagated
contractions has been reported in patients with IBS (Kellow & Phillips,
1987). They also experience pain with irregular contractions more often
than healthy persons (Kellow & Phillips, 1987; Horwitz & Fisher, 2001)

[0253] These motility findings do not account for the entire symptom
complex in patients with IBS; in fact, most of these patients do not have
demonstrable abnormalities (Rothstein R D, 2000). Patients with IBS have
increased sensitivity to visceral pain. Studies involving balloon
distention of the rectosigmoid colon have shown that patients with IBS
experience pain and bloating at pressures and volumes much lower than
control subjects (Whitehead et al., 1990). These patients maintain normal
perception of somatic stimuli.

[0254] Multiple theories have been proposed to explain this phenomenon.
For example, receptors in the viscera may have increased sensitivity in
response to distention or intraluminal contents. Neurons in the dorsal
horn of the spinal cord may have increased excitability. In addition,
alteration in CNS processing of sensations may be involved (Drossman et
al., 1997). Functional magnetic resonance imaging studies have recently
shown that compared with control subjects, patients with IBS have
increased activation of the anterior cingulate cortex, an important pain
center, in response to a painful rectal stimulus (Mertz et al., 2000).

[0255] Increasingly, evidence suggests a relationship between infectious
enteritis and subsequent development of IBS. Inflammatory cytokines may
play a role. In a survey of patients with a history of confirmed
bacterial gastroenteritis (Neal et al., 1997), 25% reported persistent
alteration of bowel habits. Persistence of symptoms may be due to
psychologic stress at the time of acute infection (Gwee et al., 1999).

[0256] Recent data suggest that bacterial overgrowth in the small
intestine may have a role in IBS symptoms. In one study (Pimentel et al.,
2000), 157 (78%) of 202 IBS patients referred for hydrogen breath testing
had test findings that were positive for bacterial overgrowth. Of the 47
subjects who had follow-up testing, 25 (53%) reported improvement in
symptoms (ie, abdominal pain and diarrhea) with antibiotic treatment.

[0257] IBS may present with a range of symptoms. However, abdominal pain
and altered bowel habits remain the primary features. Abdominal
discomfort is often described as crampy in nature and located in the left
lower quadrant, although the severity and location can differ greatly.
Patients may report diarrhea, constipation, or alternating episodes of
diarrhea and constipation. Diarrheal symptoms are typically described as
small-volume, loose stools, and stool is sometimes accompanied by mucus
discharge. Patients also may report bloating, fecal urgency, incomplete
evacuation, and abdominal distention. Upper gastrointestinal symptoms,
such as gastroesophageal reflux, dyspepsia, or nausea, may also be
present (Lynn & Friedman, 1993).

[0258] Persistence of symptoms is not an indication for further testing;
it is a characteristic of IBS and is itself an expected symptom of the
syndrome. More extensive diagnostic evaluation is indicated in patients
whose symptoms are worsening or changing. Indications for further testing
also include presence of alarm symptoms, onset of symptoms after age 50,
and a family history of colon cancer. Tests may include colonoscopy,
computed tomography of the abdomen and pelvis, and barium studies of the
small or large intestine.

[0259] L. Juvenile Rheumatoid Arthritis

[0260] `Juvenile rheumatoid arthritis` (JRA), a term for the most
prevalent form of arthritis in children, is applied to a family of
illnesses characterized by chronic inflammation and hypertrophy of the
synovial membranes. The term overlaps, but is not completely synonymous,
with the family of illnesses referred to as juvenile chronic arthritis
and/or juvenile idiopathic arthritis in Europe.

[0261] Jarvis (1998) and others (Arend, 2001) have proposed that the
pathogenesis of rheumatoid disease in adults and children involves
complex interactions between innate and adaptive immunity. This
complexity lies at the core of the difficulty of unraveling disease
pathogenesis.

[0262] Both innate and adaptive immune systems use multiple cell types, a
vast array of cell surface and secreted proteins, and interconnected
networks of positive and negative feedback (Lo et al., 1999).
Furthermore, while separable in thought, the innate and adaptive wings of
the immune system are functionally intersected (Fearon & Locksley, 1996),
and pathologic events occurring at these intersecting points are likely
to be highly relevant to our understanding of pathogenesis of adult and
childhood forms of chronic arthritis (Warrington, et al., 2001).

[0263] Polyarticular JRA is a distinct clinical subtype characterized by
inflammation and synovial proliferation in multiple joints (four or
more), including the small joints of the hands (Jarvis, 2002). This
subtype of JRA may be severe, because of both its multiple joint
involvement and its capacity to progress rapidly over time. Although
clinically distinct, polyarticular JRA is not homogeneous, and patients
vary in disease manifestations, age of onset, prognosis, and therapeutic
response. These differences very likely reflect a spectrum of variation
in the nature of the immune and inflammatory attack that can occur in
this disease (Jarvis, 1998).

[0264] M. Sjogren's Syndrome

[0265] Primary Sjogren's syndrome (SS) is a chronic, slowly progressive,
systemic autoimmune disease, which affects predominantly middle-aged
women (female-to-male ratio 9:1), although it can be seen in all ages
including childhood (Jonsson et al., 2002). It is characterized by
lymphocytic infiltration and destruction of the exocrine glands, which
are infiltrated by mononuclear cells including CD4+, CD8+ lymphocytes and
B-cells (Jonsson et al., 2002). In addition, extraglandular (systemic)
manifestations are seen in one-third of patients (Jonsson et al., 2001).

[0266] The glandular lymphocytic infiltration is a progressive feature
(Jonsson et al., 1993), which, when extensive, may replace large portions
of the organs. Interestingly, the glandular infiltrates in some patients
closely resemble ectopic lymphoid microstructures in the salivary glands
(denoted as ectopic germinal centers) (Salomonsson et al., 2002; Xanthou
& Polihronis, 2001). In SS, ectopic GCs are defined as T and B cell
aggregates of proliferating cells with a network of follicular dendritic
cells and activated endothelial cells. These GC-like structures formed
within the target tissue also portray functional properties with
production of autoantibodies (anti-Ro/SSA and anti-La/SSB) (Salomonsson
&, Jonsson, 2003).

[0267] In other systemic autoimmune diseases, such as RA, factors critical
for ectopic GCs have been identified. Rheumatoid synovial tissues with
GCs were shown to produce chemokines CXCL13, CCL21 and lymphotoxin
(LT)-13 (detected on follicular center and mantle zone B cells).
Multivariate regression analysis of these analytes identified CXCL13 and
LT-0 as the solitary cytokines predicting GCs in rheumatoid synovitis
(Weyand & Goronzy, 2003). Recently CXCL13 and CXCR5 in salivary glands
has been shown to play an essential role in the inflammatory process by
recruiting B and T cells, therefore contributing to lymphoid neogenesis
and ectopic GC formation in SS (Salomonsson & Larsson, 2002).

[0268] N. Early Arthritis

[0269] The clinical presentation of different inflammatory arthropathies
is similar early in the course of disease. As a result, it is often
difficult to distinguish patients who are at risk of developing the
severe and persistent synovitis that leads to erosive joint damage from
those whose arthritis is more self-limited. Such distinction is critical
in order to target therapy appropriately, treating aggressively those
with erosive disease and avoiding unnecessary toxicity in patients with
more self-limited disease. Current clinical criteria for diagnosing
erosive arthropathies such as rheumatoid arthritis (RA) are less
effective in early disease and traditional markers of disease activity
such as joint counts and acute phase response do not adequately identify
patients likely to have poor outcomes (Harrison & Symmons et al., 1998).
Parameters reflective of the pathologic events occurring in the synovium
are most likely to be of significant prognostic value.

[0270] Recent efforts to identify predictors of poor outcome in early
inflammatory arthritis have identified the presence of RA specific
autoantibodies, in particular antibodies towards citrullinated peptides,
to be associated with erosive and persistent disease in early
inflammatory arthritis cohorts. On the basis of this, a cyclical
citrullinated peptide (CCP) has been developed to assist in the
identification of anti-CCP antibodies in patient sera. Using this
approach, the presence of anti-CCP antibodies has been shown to be
specific and sensitive for RA, can distinguish RA from other
arthropathies, and can potentially predict persistent, erosive synovitis
before these outcomes become clinically manifest (Schellekens et al.,
2000). Importantly, anti-CCP antibodies are often detectable in sera many
years prior to clinical symptoms suggesting that they may be reflective
of subclinical immune events ((Nielen et al., 2004; Rantapaa-Dahlqvist et
al., 2003).

[0271] The clinical presentation of different inflammatory arthropathies
is similar early in the course of disease. As a result, it is often
difficult to distinguish patients who are at risk of developing the
severe and persistent synovitis that leads to erosive joint damage from
those whose arthritis is more self-limited. Such distinction is critical
in order to target therapy appropriately, treating aggressively those
with erosive disease and avoiding unnecessary toxicity in patients with
more self-limited disease. Current clinical criteria for diagnosing
erosive arthropathies such as rheumatoid arthritis (RA) are less
effective in early disease and traditional markers of disease activity
such as joint counts and acute phase response do not adequately identify
patients likely to have poor outcomes (Harrison et al., 1998). Parameters
reflective of the pathologic events occurring in the synovium are most
likely to be of significant prognostic value.

[0272] Recent efforts to identify predictors of poor outcome in early
inflammatory arthritis have identified the presence of RA specific
autoantibodies, in particular antibodies towards citrullinated peptides,
to be associated with erosive and persistent disease in early
inflammatory arthritis cohorts. On the basis of this, a cyclical
citrullinated peptide (CCP) has been developed to assist in the
identification of anti-CCP antibodies in patient sera. Using this
approach, the presence of anti-CCP antibodies has been shown to be
specific and sensitive for RA, can distinguish RA from other
arthropathies, and can potentially predict persistent, erosive synovitis
before these outcomes become clinically manifest. Importantly, anti-CCP
antibodies are often detectable in sera many years prior to clinical
symptoms suggesting that they may be reflective of subclinical immune
events (Nielen et al., 2004; Rantapaa-Dahlqvist et al., 2003).

[0273] O. Psoriasis

[0274] Psoriasis is a chronic skin disease of scaling and inflammation
that affects 2 to 2.6 percent of the United States population, or between
5.8 and 7.5 million people. Although the disease occurs in all age
groups, it primarily affects adults. It appears about equally in males
and females. Psoriasis occurs when skin cells quickly rise from their
origin below the surface of the skin and pile up on the surface before
they have a chance to mature. Usually this movement (also called
turnover) takes about a month, but in psoriasis it may occur in only a
few days. In its typical form, psoriasis results in patches of thick, red
(inflamed) skin covered with silvery scales. These patches, which are
sometimes referred to as plaques, usually itch or feel sore. They most
often occur on the elbows, knees, other parts of the legs, scalp, lower
back, face, palms, and soles of the feet, but they can occur on skin
anywhere on the body. The disease may also affect the fingernails, the
toenails, and the soft tissues of the genitals and inside the mouth.
While it is not unusual for the skin around affected joints to crack,
approximately 1 million people with psoriasis experience joint
inflammation that produces symptoms of arthritis. This condition is
called psoriatic arthritis.

[0275] Psoriasis is a skin disorder driven by the immune system,
especially involving a type of white blood cell called a T cell.
Normally, T cells help protect the body against infection and disease. In
the case of psoriasis, T cells are put into action by mistake and become
so active that they trigger other immune responses, which lead to
inflammation and to rapid turnover of skin cells. In about one-third of
the cases, there is a family history of psoriasis. Researchers have
studied a large number of families affected by psoriasis and identified
genes linked to the disease. People with psoriasis may notice that there
are times when their skin worsens, then improves. Conditions that may
cause flareups include infections, stress, and changes in climate that
dry the skin. Also, certain medicines, including lithium and
betablockers, which are prescribed for high blood pressure, may trigger
an outbreak or worsen the disease.

[0276] P. Neuroinflammation

[0277] Neuroinflammation encapsulates the idea that microglial and
astrocytic responses and actions in the central nervous system have a
fundamentally inflammation-like character, and that these responses are
central to the pathogenesis and progression of a wide variety of
neurological disorders. This idea originated in the field of Alzheimer's
disease (Griffin et al., 1989; Rogers et al., 1988), where it has
revolutionized our understanding of this disease (Akiyama et al., 2000).
These ideas have been extended to other neurodegenerative diseases
(Eikelenboom et al., 2002; Orr et al., 2002; Ishizawa & Dickson, 2001),
to ischemic/toxic diseases (Gehrmann et al., 1995; Touzani et al., 1999),
to tumor biology (Graeber et al., 2002) and even to normal brain
development.

[0278] Neuroinflammation incorporates a wide spectrum of complex cellular
responses that include activation of microglia and astrocytes and
induction of cytokines, chemokines, complement proteins, acute phase
proteins, oxidative injury, and related molecular processes. These events
may have detrimental effects on neuronal function, leading to neuronal
injury, further glial activation, and ultimately neurodegeneration.

[0279] Neuroinflammation is a new and rapidly expanding field that has
revolutionized our understanding of chronic neurological diseases. This
field encompasses research ranging from population studies to signal
transduction pathways, and investigators with backgrounds in fields as
diverse as pathology, biochemistry, molecular biology, genetics, clinical
medicine, and epidemiology. Important contributions to this field have
come from work with populations, with patients, with postmortem tissues,
with animal models, and with in vitro systems.

III. CYTOKINE ASSAYS

[0280] Traditional cytokine assays in involve measurement of cytokines in
serum or plasma, but various cytokines have been detected in other
biological fluids. For example, Kimball (1984) reported IL-1 bioactivity
in human urine. Tamatani et al. (1988) disclosed the presence of
IL-1α and IL-1β in human amniotic fluid, using chromatographic
and bioassay methods. The same group used enzyme immunoassays to measure
IL-1α and IL-1β in human amniotic fluid (Tsunoda et. al.,
1988). Wilmott et al. (1988) measured IL-1β and IL-1 bioactivity in
human bronchoalveolar lavage fluid in cystic fibrosis compared to other
diseases. Khan et al. (1988) reported that high levels of IL-1-like
bioactivity could be demonstrated in human ovarian follicular fluid.
Lymphotoxins have been reported in blister fluid of pemphigoid patients
(Jeffes et al., 1984). IL-1 has also been reported in human sweat
(Didierjean et al., 1990). IL-1 has been reported to be found in the
cerebrospinal fluid (CSF) of cats (Coceani et al., 1988) and humans (see,
for example, Peter et al., 1991). A factor with IL-1-like bioactivity was
detected in the gingival fluid of clinically normal humans (Oppenheim et
al., 1982), the activity being higher in inflamed than non-inflamed
gingival regions.

[0281] Other methods can be used to quantify specific cytokine expression
including methods that measure cytokine mRNA or cytokine polypeptide. For
example, PCR®, competitive PCR®, PCR-ELISA, Microarrays, gene
expression bead-based assays, and in situ hybridization techniques can be
used to measure cytokine mRNA, and immunohistochemistry can be used to
measure cytokine protein levels.

[0282] A. Genetic Assays

[0283] One embodiment of the instant invention comprises a method for
detecting variation in cytokine levels using nucleic acid based studies.
The nucleic acid is isolated from cells contained in any appropriate
biological sample, according to standard methodologies (Sambrook et al.,
1989). The nucleic acid may be genomic DNA or fractionated or whole cell
RNA. Where RNA is used, it may be desired to convert the RNA to a
complementary DNA. In one embodiment, the RNA is whole cell RNA; in
another, it is poly-A RNA. Normally, the nucleic acid is amplified.

[0284] Depending on the format, the specific nucleic acid of interest is
identified in the sample directly using amplification or with a second,
known nucleic acid following amplification. Next, the identified product
is detected. In certain applications, the detection may be performed by
visual means (e.g., ethidium bromide staining of a gel). Alternatively,
the detection may involve indirect identification of the product via
chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent
label or even via a system using electrical or thermal impulse signals
(Affymax Technology; Bellus, 1994).

[0285] Following detection, one may compare the results seen in a given
patient with a statistically significant reference group of normal
patients and patients that have cytokine-related pathologies. In this
way, it is possible to correlate the amount or kind of cytokines detected
with various clinical states.

[0286] i. Southern/Northern Blotting

[0287] Blotting techniques are well known to those of skill in the art.
Southern blotting involves the use of DNA as a target, whereas Northern
blotting involves the use of RNA as a target. Each provide different
types of information, although cDNA blotting is analogous, in many
aspects, to blotting or RNA species.

[0288] Briefly, a probe is used to target a DNA or RNA species that has
been immobilized on a suitable matrix, often a filter of nitrocellulose.
The different species should be spatially separated to facilitate
analysis. This often is accomplished by gel electrophoresis of nucleic
acid species followed by "blotting" on to the filter.

[0289] Subsequently, the blotted target is incubated with a probe (usually
labeled) under conditions that promote denaturation and rehybridization.
Because the probe is designed to base pair with the target, the probe
will binding a portion of the target sequence under renaturing
conditions. Unbound probe is then removed, and detection is accomplished
as described above.

[0290] ii. Separation Methods

[0291] It normally is desirable, at one stage or another, to separate the
amplification product from the template and the excess primer for the
purpose of determining whether specific amplification has occurred. In
one embodiment, amplification products are separated by agarose,
agarose-acrylamide or polyacrylamide gel electrophoresis using standard
methods. See Sambrook et al., 1989.

[0292] Alternatively, chromatographic techniques may be employed to effect
separation. There are many kinds of chromatography which may be used in
the present invention: adsorption, partition, ion-exchange and molecular
sieve, and many specialized techniques for using them including column,
paper, thin-layer and gas chromatography (Freifelder, 1982).

Detection Methods

[0293] Products may be visualized in order to confirm amplification of the
marker sequences. One typical visualization method involves staining of a
gel with ethidium bromide and visualization under UV light.
Alternatively, if the amplification products are integrally labeled with
radio- or fluorometrically-labeled nucleotides, the amplification
products can then be exposed to x-ray film or visualized under the
appropriate stimulating spectra, following separation.

[0294] iii. Kit Components

[0295] All the essential materials and reagents required for detecting and
sequencing cytokines may be assembled together in a kit. This generally
will comprise preselected primers and probes. Also included may be
enzymes suitable for amplifying nucleic acids including various
polymerases (RT, Taq, Sequenase®, etc.), deoxynucleotides and buffers
to provide the necessary reaction mixture for amplification. Such kits
also generally will comprise, in suitable means, distinct containers for
each individual reagent and enzyme as well as for each primer or probe.

[0296] iv. RT-PCRT® (Relative Quantitative)

[0297] Reverse transcription (RT) of RNA to cDNA followed by relative
quantitative PCR® (RT-PCRT®) can be used to determine the relative
concentrations of specific mRNA species isolated from patients. By
determining that the concentration of a specific mRNA species varies, it
is shown that the gene encoding the specific mRNA species is
differentially expressed.

[0298] In PCR®, the number of molecules of the amplified target DNA
increase by a factor approaching two with every cycle of the reaction
until some reagent becomes limiting. Thereafter, the rate of
amplification becomes increasingly diminished until there is no increase
in the amplified target between cycles. If a graph is plotted in which
the cycle number is on the X axis and the log of the concentration of the
amplified target DNA is on the Y axis, a curved line of characteristic
shape is formed by connecting the plotted points. Beginning with the
first cycle, the slope of the line is positive and constant. This is said
to be the linear portion of the curve. After a reagent becomes limiting,
the slope of the line begins to decrease and eventually becomes zero. At
this point the concentration of the amplified target DNA becomes
asymptotic to some fixed value. This is said to be the plateau portion of
the curve.

[0299] The concentration of the target DNA in the linear portion of the
PCR® amplification is directly proportional to the starting
concentration of the target before the reaction began. By determining the
concentration of the amplified products of the target DNA in PCR®
reactions that have completed the same number of cycles and are in their
linear ranges, it is possible to determine the relative concentrations of
the specific target sequence in the original DNA mixture. If the DNA
mixtures are cDNAs synthesized from RNAs isolated from different tissues
or cells, the relative abundances of the specific mRNA from which the
target sequence was derived can be determined for the respective tissues
or cells. This direct proportionality between the concentration of the
PCR® products and the relative mRNA abundances is only true in the
linear range of the PCR® reaction.

[0300] The final concentration of the target DNA in the plateau portion of
the curve is determined by the availability of reagents in the reaction
mix and is independent of the original concentration of target DNA.
Therefore, the first condition that must be met before the relative
abundances of a mRNA species can be determined by RT-PCR® for a
collection of RNA populations is that the concentrations of the amplified
PCR® products must be sampled when the PCR® reactions are in the
linear portion of their curves.

[0301] The second condition that must be met for an RT-PCR® experiment
to successfully determine the relative abundances of a particular mRNA
species is that relative concentrations of the amplifiable cDNAs must be
normalized to some independent standard. The goal of an RT-PCR®
experiment is to determine the abundance of a particular mRNA species
relative to the average abundance of all mRNA species in the sample. In
the experiments described below, mRNAs for β-actin, asparagine
synthetase and lipocortin II were used as external and internal standards
to which the relative abundance of other mRNAs are compared.

[0302] Most protocols for competitive PCR® utilize internal PCR®
standards that are approximately as abundant as the target. These
strategies are effective if the products of the PCR® amplifications
are sampled during their linear phases. If the products are sampled when
the reactions are approaching the plateau phase, then the less abundant
product becomes relatively over represented. Comparisons of relative
abundancies made for many different RNA samples, such as is the case when
examining RNA samples for differential expression, become distorted in
such a way as to make differences in relative abundances of RNAs appear
less than they actually are. This is not a significant problem if the
internal standard is much more abundant than the target. If the internal
standard is more abundant than the target, then direct linear comparisons
can be made between RNA samples.

[0303] The above discussion describes theoretical considerations for an
RT-PCR® assay for clinically derived materials. The problems inherent
in clinical samples are that they are of variable quantity (making
normalization problematic), and that they are of variable quality
(necessitating the co-amplification of a reliable internal control,
preferably of larger size than the target). Both of these problems are
overcome if the RT-PCR® is performed as a relative quantitative
RT-PCR® with an internal standard in which the internal standard is an
amplifiable cDNA fragment that is larger than the target cDNA fragment
and in which the abundance of the mRNA encoding the internal standard is
roughly 5-100 fold higher than the mRNA encoding the target. This assay
measures relative abundance, not absolute abundance of the respective
mRNA species.

[0304] Other studies may be performed using a more conventional relative
quantitative RT-PCR® assay with an external standard protocol. These
assays sample the PCR® products in the linear portion of their
amplification curves. The number of PCR® cycles that are optimal for
sampling must be empirically determined for each target cDNA fragment. In
addition, the reverse transcriptase products of each RNA population
isolated from the various tissue samples must be carefully normalized for
equal concentrations of amplifiable cDNAs. This consideration is very
important since the assay measures absolute mRNA abundance. Absolute mRNA
abundance can be used as a measure of differential gene expression only
in normalized samples. While empirical determination of the linear range
of the amplification curve and normalization of cDNA preparations are
tedious and time consuming processes, the resulting RT-PCR® assays can
be superior to those derived from the relative quantitative RT-PCR®
assay with an internal standard.

[0305] One reason for this advantage is that without the internal
standard/competitor, all of the reagents can be converted into a single
PCR® product in the linear range of the amplification curve, thus
increasing the sensitivity of the assay. Another reason is that with only
one PCR® product, display of the product on an electrophoretic gel or
another display method becomes less complex, has less background and is
easier to interpret.

[0306] Still other studies may be performed using "real-time" RT-PCR®
(Higuchi et al., 1993). These assays detect PCR® products as they
accumulate instead of detecting the amount of PCR® products
accumulated after a fixed number of cycles. A method of detecting
fluorescence after each PCR® cycle is required. The fluorescence
signal is plotted versus the cycle number. The cycle number is expressed
as the threshold cycle (CT). The initial fluorescence defines the
baseline for the plot and an accumulated PCR® product is indicated by
an increase in fluorescence above the baseline. Quantification of the
amount of target in a sample is determined by measuring and comparing the
CT to a standard curve to determine the starting copy number.

[0307] "Real-Time" RT-PCR® (Higuchi et al., 1993) provides more precise
quantitation of the amount of target because it is determined during the
exponential phase of PCR®, rather than at the endpoint. It also allows
higher throughput because the use of CT values allow a larger
dynamic range. Dilutions of each sample are no longer required.

[0308] v. Gene Expression Bead-Based Assay

[0309] A gene expression bead-based assay a protocol was created to show
oligonucleotide probes could be attached on the surface with a density
optimally suited for gene expression measurement. The attachment method
(creating the covalent bond between the carboxylated bead surface and the
amine linked oligonucleotide probe, utilizing a carbodiimide-activated
succinimide coupling chemistry) was verified with a special 40-mer
oligonucleotide probe linked at the 5' end with an amine linker molecule
and at the 3' end with a biotin group. The reporter of the fluorescent
signal is a streptavidin-linked R-PE (R-Phycoerythrin) complex that
produces signal based on the presence of biotin groups at the bead
surface. By using this special sequence no hybridization was necessary to
verify the oligo probe coupling protocol. Additionally, the amount of
oligo probe added to the bead-coupling reaction was titrated through two
orders of magnetude to gain a sense of how the surface density of
attachment affects fluorescent signal.

[0310] This technique enables us to attach any short oligonucleotide
sequence to the bead surface to serve as probe in hybridization
reactions.

[0311] The gene expression assay was initially tested with optimum
conditions by using an HPLC-purified anti-sense 70-mer target with a
biotinylated 5' end in the hybridization reaction and no other
competitive sequences. The probes used were selected from the 70mer
oligonucleotide library used in our microarray core facility. The
hybridization reactions were designed to vary both time and amount of
target complement in order to generate rate of reaction graphs and
concentration-response curves, respectively. Additionally,
cross-reactivity was shown to be under 8% (most data points were under 5%
(even under non-stringent conditions at 37° C.) to demonstrate
sequence specificity for the bead-based system.

[0312] Tests with cDNA transcribed from commercially available,
predictably performing mRNA was first used to assess efficacy of the
overall bead-based gene expression process. Using oligo-dT as a primer
and biotinylated-dUTP to directly label the cDNA transcript,
concentration dependant signal was assessed to create a calibration curve
for the assay. This process was verified using both human and mouse RNA.
To cross-validate the data, and to demonstrate the scalability from
microarray platform to bead platform, particular probes chosen from
microarray experiments were tested with the same pool of mouse RNA
harvested from brain and liver sources. These particular probes were
selected from the microarray experiments using the superior data mining
techniques developed in our laboratory's bioinformatics core and were
shown to be biologically unique and relevant to a particular tissue type
(either brain or liver). One hundred percent of the probes assayed showed
directional expression matches and concurred with QT-PCR measurements
(Table 1) providing powerful evidence of the potential to streamline the
costly, labor intensive microarray data onto a smaller, higher-sample
throughput, more cost effective assay platform.

[0313] The significance of this approach is further augmented by the
powerful clinical need for rapid, inexpensive multi-biomarker diagnostic
assays requiring a thorough understanding of three main areas of
research: protocol design, biosensing paradigm, and lab automation
strategy.

[0314] Screening of genome-scale microarrays provides the best means of
rapidly identifying gene-expression based biomarkers in preparation for
next generation clinical assays. Once the previously unknown sequences of
diagnostic and therapeutic relevance have been identified from samples
the results will be immediately translated to the bead-based assay
platform. Becaucse sequence specificity is the primary force behind
specificity and signal, oligonucleotides of the exact same length and
sequence used in the microarray studies are chosen for attachment to the
carboxylated bead surface to mimic the hybridization chemistry on the
microarray platform. These oligonucleotides are prepared with a 12-carbon
spacer at the 5'-end. The attachment is achieved by using an organic
linker at pH 4.5 to create the conditions for hydrolysis thereby creating
the covalent bond between the amine and carboxylic acid groups. This
enables the oligonucleotides to be permanently attached to the 5.6-micron
diameter polystyrene beads and stored safely for up to one year at
4° C. in the stabilizing buffer (TE pH 8.0).

[0315] Oligo probe attachment is verified with same length reverse
complement sequences that have a biotinylated 5' end. If the probes have
been successfully attached the signals will correlate directly to the
amount of reverse complement sequence added. The signals are acquired via
a streptavidin-phycoerythrin label that attaches itself to the
biotinylated end of the reverse complement. The R-PE fluorescent signals
are generated by 532 nm 10 mW laser and amplified with a 14-bit A/D PMT
(analog to digital photomultiplier tube) for digital signal processing.
The signals generated are dose dependant and serve to create a
calibration curve to track variance from lot to lot of prepared beads.

[0316] By way of example, peripheral blood mononuclear cells (PBMCs) were
collected from rheumatoid arthritis patients at several stages of disease
severity and undergoing pleural therapeutic treatment. The cellular mRNA
was extracted using the RNAeasy Kit from Qiagen. cDNA is synthesized
using a reverse transcriptase enzyme provided by Qiagen (Omniscript).
There is no amplification of the product through multiple cycles; the
cDNA is the result of a first strand synthesis. The label, bioin-16-dUTP,
is directly incorporated into the cDNA during synthesis using a 3.0/2.5
ratio of label to dTTP. The reverse transcriptase is superior to those
often used in the microarray scientific community due to its ability to
prevent random priming. However, the removal of cDNA created from random
priming results in an attenuation of fluorescent signal due to overall
lower transcript abundance. The prevention of this enzymatic effect
during the cDNA synthesis step allowed for our never before seen 100%
validation of signal ratios via QT-PCR.

[0317] The molar ratio of 3.0/2.5 was successful in previous research
studies used to validate the bead-based gene expression system. The
strategy employed to determine incorporation efficiency will involve
spotting a serial dilution of cDNA product (created with several
different molar ratios of label) onto a nitrocellulose membrane to
participate in a horseradish peroxidase-flourchrome mediated luminescence
reaction. This method will enable us to demonstrate the successful
creation of cDNA and document the influence of any change in synthesis
conditions on cDNA production (such as using gene specific primers,
random primers, oligo-dT primers, or biotinylated oligo-dT primers).

[0318] Hybridization conditions for the bead-based assay are carefully
selected due to the mobility of the reaction surface (the beads move
freely in the solution and not attached to any surface). Temperature and
hybridization time affect not only the rate of reaction, but also the
specificity of the signals generated. In order to generate signals of
sufficient specificity we utilize a 5 molar TMAC (Tetramethyl-amonium
chloride, Sigma) to create high-salt stringency conditions. The critical
chemical property of TMAC is its ability to nearly equalize the bond
strength between A-T/G-C dsDNA bonds creating a uniform Tm between the
competing probes bound to serparate bead types in the multiplexed
reaction. The high temperature of 58° C. is chosen also for
stringency reasons, and is based on the theoretical Tm of a 70-mer
oligonucleotide in 5 normal TMAC. The driving force behind the buffer is
to create a sequence specific exclusion reaction at a single temperature,
and although creating high stringency conditions inevitably results in an
attenuation of fluorescent signal, these conditions are necessary to
generate diagnostically relevant results.

[0319] Keeping the beads in suspension during the hybridization is
necessary to achieve equal accessibility of target to probe at the bead
surface. High salt concentration conditions raise the density of the
solution above that of polystyrene (the bead composition). This causes
the beads to float in static conditions, changing the surface
availability for the reaction, potentially lowering the sensitivity of
the gene expression measurement. Fortunately the difference in density is
small and gentle mixing of many types can keep the beads suspended. This
environment has been simulated by using a hybridization oven (Hybaid at
58° C.) capable of rotating the sealed reaction tubes at 10 rpm
yielding good results. However, these conditions are a challenge for
automation and high throughput, which is why we are developing solutions
to integrate the ideal hybridization conditions with the Biomek FX lab
automation system.

[0320] Even though the hybridization for the bead-based assay has been
tailored to mimic the microarray environment as closely as possible to
generate the environment to easily scale from one assay to the next,
there are subtle differences that mandate that well designed internal
controls are a necessity for this project. The normalization and scaling
methods for the microarray community rely upon data gathered from
thousands of spots on the array, but the bead-based assay can measure at
most 100 different transcripts automatically making the normalization and
scaling methods different from the microarray.

[0321] The positive control is using plant RNA from A. thaliana, the
`RUBISCO` gene (GenBank ID: X14212). By using a known amount of
sequence-dissimilar RNA spiked into the cDNA synthesis step, we can get a
more clear idea of the general success of the labeling step. Furthermore,
beads with the appropriate plant probe sequence help describe the complex
relationship between starting mRNA concentration and fluorescent signal
generation.

[0322] The negative control is designed around random 70mer sequences
(with no sequence specificity in the human genome) attached to the bead
surface, which in theory could not possibly have any target sequences
able to bind to them. Any fluorescent signal given off by these beads
would provide a measure of background noise produced by
cross-hybridization. These fluorescent signals will be used to normalize
the patient sample signals to a common background making the inter-well
comparisons straightforward.

[0323] The most academically accepted way to demonstrate scalability from
the microarray platform is to have fluorescent ratios from both systems
agree with QT-PCR cross-validation experiments and match directionally.
This cross-validation will serve to strengthen the argument that all
systems are measuring gene expression in tandem, and differentially
between two samples.

[0324] Utilizing the Beckman-Coulter Biomek FK system, an automated
process to perform the gene-expression bead-based assay has been
developed. The Biomek FX can easily integrate with a thermocycling device
and various heat blocks to make cDNA synthesis and hybridization
conditions automation friendly.

[0325] The R-PE molecule is stable at hybridization pH (8.2) and
temperature (58° C.), but only by a narrow margin (stability is
lost at 60° C.). Since proper washing has removed
cross-hybridizing sequences, it is safe to perform this step across a
wide range of temperatures. We have seen no difference in using
37° C. versus hybridization temperature at this final labeling
step for reverse complement sequences, allowing flexibility in post
hybridization labeling strategies.

[0326] The final fluorescent signal is created via phycoerythrin, a high
quantum yield protein produced by red algae. This protein is covalently
linked to streptavidin making the biotinylated nucleotides incorporated
into the cDNA accessible to this fluorescent label. The fluorescent
signals are measured with the same device as the ICMP panel: a 532 nm
laser excites the phycoerythrin, the light produced is amplified and
digitized by a PMT and filtered by a digital signal processor before
being sent to the PC computer for storage and analysis.

[0327] B. Immunodiagnosis

[0328] Antibodies (discussed above) of the present invention can be used
in characterizing the cytokine content of various biological samples
through techniques such as ELISAs and Western blotting. This may provide
a screen for the presence or absence of malignancy or as a predictor of
future cancer.

[0329] The use of antibodies in an ELISA assay is specifically
contemplated. For example, anti-cytokine antibodies are immobilized onto
a selected surface, preferably a surface exhibiting a protein affinity
such as the wells of a polystyrene microtiter plate. After washing to
remove incompletely adsorbed material, it is desirable to bind or coat
the assay plate wells with a non-specific protein that is known to be
antigenically neutral with regard to the test antisera such as bovine
serum albumin (BSA), casein or solutions of powdered milk. This allows
for blocking of non-specific adsorption sites on the immobilizing surface
and thus reduces the background caused by non-specific binding of antigen
onto the surface.

[0330] After binding of antibody to the well, coating with a non-reactive
material to reduce background, and washing to remove unbound material,
the immobilizing surface is contacted with the sample to be tested in a
manner conducive to immune complex (antigen/antibody) formation.
Following formation of specific immunocomplexes between the test sample
and the bound antibody, and subsequent washing, the occurrence and even
amount of immunocomplex formation may be determined by subjecting same to
a second antibody having specificity for a cytokine that differs from the
first antibody. Appropriate conditions preferably include diluting the
sample with diluents such as BSA, bovine gamma globulin (BGG) and
phosphate buffered saline (PBS)/Tween®. These added agents also tend
to assist in the reduction of nonspecific background. The layered
antisera is then allowed to incubate for from about 2 to about 4 hr, at
temperatures preferably on the order of about 25° to about
27° C. Following incubation, the antisera-contacted surface is
washed so as to remove non-immunocomplexed material. A preferred washing
procedure includes washing with a solution such as PBS/Tween®, or
borate buffer.

[0331] To provide a detecting means, the second antibody will preferably
have an associated enzyme that will generate a color development upon
incubating with an appropriate chromogenic substrate. Thus, for example,
one will desire to contact and incubate the second antibody-bound surface
with a urease, alkaline phosphatase, glucose oxidase, or (horseradish)
peroxidase-conjugated anti-human IgG for a period of time and under
conditions which favor the development of immunocomplex formation (e.g.,
incubation for 2 hr at room temperature in a PBS-containing solution such
as PBS/Tween®).

[0332] After incubation with the second enzyme-tagged antibody, and
subsequent to washing to remove unbound material, the amount of label is
quantified by incubation with a chromogenic substrate such as urea and
bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic
acid (ABTS) and H2O2, in the case of peroxidase as the enzyme
label. Quantitation is then achieved by measuring the degree of color
generation, e.g., using a visible spectrum spectrophotometer.

[0333] The preceding format may be altered by first binding the sample to
the assay plate. Then, primary antibody is incubated with the assay
plate, followed by detecting of bound primary antibody using a labeled
second antibody with specificity for the primary antibody.

[0334] The antibody compositions of the present invention will find great
use in immunoblot or Western blot analysis. The antibodies may be used as
high-affinity primary reagents for the identification of proteins
immobilized onto a solid support matrix, such as nitrocellulose, nylon or
combinations thereof. In conjunction with immunoprecipitation, followed
by gel electrophoresis, these may be used as a single step reagent for
use in detecting antigens against which secondary reagents used in the
detection of the antigen cause an adverse background.
Immunologically-based detection methods for use in conjunction with
Western blotting include enzymatically-, radiolabel-, or
fluorescently-tagged secondary antibodies against the toxin moiety are
considered to be of particular use in this regard.

[0335] The antibodies of the present invention may also be used in
conjunction with both fresh-frozen and/or formalin-fixed,
paraffin-embedded tissue blocks, such as blocks prepared from a tumor
biopsy, prepared for study by immunohistochemistry (NC). The method of
preparing tissue blocks from these particulate specimens has been
successfully used in previous NC studies of various prognostic factors,
and/or is well known to those of skill in the art (Brown et cd., 1990;
Abbondanzo et al., 1999; Allred et al., 1990).

[0337] Permanent-sections may be prepared by a similar method involving
rehydration of the 50 mg sample in a plastic microfuge tube; pelleting;
resuspending in 10% formalin for 4 hours fixation; washing/pelleting;
resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden
the agar; removing the tissue/agar block from the tube; infiltrating
and/or embedding the block in paraffin; and/or cutting up to 50 serial
permanent sections.

[0338] Another form of immunodiagnosis involves protein array technology,
which allows high-throughput screening. Protein arrays, in this case
populated by various anti-cytokine antibodies, appear as new and
versatile tools in functional genomics, enabling the translation of gene
expression patterns of normal and diseased tissues into protein product
catalog. These arrays amy contain thousands of different antibodies
spotted onto glass slides or immobilized in tiny wells, and allow one to
examine the level of protein expression for a large number of proteins at
once.

[0339] The basic construction of protein chips has some similarities to
DNA chips, such as the use of a glass or plastic surface dotted with an
array of molecules. These molecules can be DNA or antibodies that are
designed to capture proteins. Defined quantities of proteins are
immobilized on each spot, while retaining some activity of the protein.
With fluorescent markers or other methods of detection revealing the
spots that have captured these proteins, protein microarrays are being
used as powerful tools in high-throughput proteomics and drug discovery.

[0340] Glass slides are still widely used, since they are inexpensive and
compatible with standard microarrayer and detection equipment. However,
their limitations include multiple-based reactions, high evaporation
rates, and possible cross-contamination. Matrix slides offer a number of
advantages, such as reduced evaporation and no possibility of
cross-contamination, but they are expensive. Nanochips for proteomics
have the same advantages, in addition to reduced cost and the capability
of multiple-component reactions.

[0341] The earliest and best-known protein chip is the ProteinChip by
Ciphergen Biosystems Inc. (Fremont, Calif.). The ProteinChip is based on
the surface-enhanced laser desorption and ionization (SELDI) process.
Known proteins are analyzed using functional assays that are on the chip.
For example, chip surfaces can contain enzymes, receptor proteins, or
antibodies that enable researchers to conduct protein-protein interaction
studies, ligand binding studies, or immunoassays. With state-of-the-art
ion optic and laser optic technologies, the ProteinChip system detects
proteins ranging from small peptides of less than 1000 Da up to proteins
of 300 kDa and calculates the mass based on time-of-flight (TOF).

[0342] The ProteinChip biomarker system is the first protein biochip-based
system that enables biomarker pattern recognition analysis to be done.
This system allows researchers to address important clinical questions by
investigating the proteome from a range of crude clinical samples (i.e.,
laser capture microdissected cells, biopsies, tissue, urine, and serum).
The system also utilizes biomarker pattern software that automates
pattern recognition-based statistical analysis methods to correlate
protein expression patterns from clinical samples with disease
phenotypes.

[0343] Some systems can perform biomarker discovery in days and validation
of large sample sets within weeks. Its robotics system accessory
automates sample processing, allowing hundreds of samples to be run per
week and enabling a sufficient number of samples to be run, which
provides high statistical confidence in comprehensive studies for marker
discovery and validation.

[0344] Microfluidics is one of the most important innovations in biochip
technology. Since microfluidic chips can be combined with mass
spectrometric analysis, a microfluidic device has been devised in which
an electrospray interface to a mass spectrometer is integrated with a
capillary electrophoresis channel, an injector, and a protein digestion
bed on a monolithic substrate (Wang et al., 2000). This chip thus
provides a convenient platform for automated sample processing in
proteomics applications.

[0345] These chips can also analyze expression levels of serum proteins
with detection limits comparable to commercial enzyme-linked
immunosorbent assays, with the advantage that the required volume sample
is markedly lower compared with conventional technologies.

[0346] Biosite (San Diego) manufactures the Triage protein chip that
simultaneously measures 100 different proteins by immunoassays. The
Triage protein chip immunoassays are performed in a microfluidic plastic
chip, and the results are achieved in 15 minutes with picomolar
sensitivities. Microfluidic fluid flow is controlled in the protein chip
by the surface architecture and surface hydrophobicity in the
microcapillaries. The immunoassays utilize high-affinity antibodies and a
near-infrared fluorescent label, which is read by a fluorometer.

[0347] C. Nanoscale Protein Analysis

[0348] Most current protocols including protein purification and automated
identification schemes yield low recoveries that limit the overall
process in terms of sensitivity and speed. Such low protein yields and
proteins that can only be isolated from limited source material (e.g.,
biopsies) can be subjected to nanoscale protein analysis: a nanocapture
of specific proteins and complexes, and optimization of all subsequent
sample-handling steps, leading to a mass analysis of peptide fragments.
This focused approach, also termed targeted proteomics, involves
examining subsets of the proteome (e.g., those proteins that are
specifically modified, bind to a particular DNA sequence, or exist as
members of higher-order complexes or any combination thereof). This
approach is used to identify genetic determinants of cancer that alter
cellular physiology and respond to agonists.

[0349] A new detection technique called multiphoton detection, by Biotrace
Inc. (Cincinnati), can quantify subzeptomole amounts of proteins and will
be used for diagnostic proteomics, particularly for cytokines and other
low-abundance proteins. Biotrace is also developing supersensitive
protein biochips to detect concentrations of proteins as low as 5 fg/ml
(0.2 attomole/ml), thereby permitting sensitivity that is 1000 times
greater than current protein biochips.

[0352] If the cytokine profiles indicate that a patient has severe
disease, then that patient also can be classified as being predisposed to
develop a severe AS condition including major organ involvement and major
joint destruction. If the cytokine profile indicates that a patient has
mild or intermediate disease, then further analysis can be performed to
determine that patient's predisposition to develop severe disease.

[0353] Specifically, the invention involves analyzing peripheral blood
serum, plasma, synovial fluid, cerebrospinal fluid, a tissue sample, or
other body fluid sample for particular cytokines such as IL-1α,
IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12,
IL-13, IL-15, IL-17, IL-18, INF-α, INF-β, INF-γ,
TNF-α, GM-CSF, G-CSF, MIP-1α/CCL3, MIP-1β/CCL4,
MCP-1/CCL2, RANTES/CCL5, Eotaxin/CCL11, VEGF, EGF, or FGF. The reference
level can be the median level of the cytokine found in samples derived
from a population. The population can include a population of patients
having ankylosing spondylitis, a population of healthy individuals, a
population of patients having extra-articular involvement, or a
population of patients having axial joint destruction.

[0354] A particular level of a particular cytokine can be determined to be
high or low based on the levels measured from various populations. Such
populations can include, without limitation, populations of patients with
ankylosing spondylitis, patients with extra-articular involvement,
patients with axial joint destruction, and healthy individuals.

[0355] The invention also is based on the discovery that patients
presenting similar AS symptoms can have different levels of particular
cytokines within their serum, plasma, synovial fluid, tissue,
cerebrospinal fluid, or other body fluid samples. Thus, determining the
peripheral blood, serum, plasma, synovial fluid, tissue, cerebrospinal
fluid, or other body fluid cytokine profile can be used to determine the
proper treatment protocol for each individual patient. For example, two
patients having similar AS symptoms may have different levels of IL-10
within their serum. The patient with low levels may benefit from a
treatment of IL-10 while the patient with high levels of IL-10 may
benefit from treatment with IL-10 inhibitors such as anti-IL-10 antibody
drugs, a class of pharmaceuticals called biological drugs. Thus,
determining the cytokine profile from a patient can help provide doctors
and patients with information that can be used to determine adequate
treatments and measure an individual patient's response to treatment.

[0356] Another embodiment of the invention features a method for
determining the predisposition of an ankylosing spondylitis patient to
develop severe disease. The method includes determining the level of a
cytokine (e.g., CCL4, CCL2, CCL11, EGF, IL-1β, IL-2, IL-5, IL-6,
IL-7, CXCL8, IL-10, IL-12, IL-13, IL-15, IL-17, TNF-α, IFNγ,
GM-CSF, or G-CSF) within a sample from the patient, comparing the level
of the cytokine to a reference level to obtain information about the
ankylosing spondylitis condition. The sample can be from peripheral blood
serum, plasma, synovial fluid, tissue biopsy (e.g., a synovial tissue
biopsy), cerebrospinal fluid, or other body fluid. The reference level
can be the median level of the cytokine found in samples derived from a
population. The population can include a population of patients having
ankylosing spondylitis, a population of healthy individuals, a population
of patients having extra-articular involvement, or a population of
patients having axial joint destruction.

[0359] If the cytokine profiles indicate that a patient has severe
disease, then that patient also can be classified as being predisposed to
develop a severe PsA condition including major organ involvement and
major joint destruction. If the cytokine profile indicates that a patient
has mild or intermediate disease, then further analysis can be performed
to determine that patient's predisposition to develop severe disease.

[0360] Specifically, the invention involves analyzing peripheral blood
serum, cerebrospinal fluid, a tissue sample, or other body fluid sample
for particular cytokines such as, IL-1α, IL-1β, IL-2, IL-4,
IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18,
INF-α, INF-β, INF-γ, TNF-α, GM-CSF, G-CSF,
MIP-1α/CCL3, MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5,
Eotaxin/CCL11, VEGF, EGF, or FGF. The reference level can be the median
level of the cytokine found in samples derived from a population. The
population can include a population of patients having a mild PsA
condition, a population of patients having a intermediate PsA condition,
a population of patients having a severe PsA condition, a population of
healthy individuals, a population of patients having extra-articular
involvement, or a population of patients having major joint destruction.

[0361] A particular level of a particular cytokine can be determined to be
high or low based on the levels measured from various populations. Such
populations can include, without limitation, populations of patents with
a mild condition, intermediate condition, or severe condition, patients
with extra-articular involvement, patients with major joint destruction,
and healthy individuals.

[0362] The invention also is based on the discovery that patients
presenting similar PsA symptoms can have different levels of particular
cytokines within their serum, tissue, cerebrospinal fluid, or other body
fluid samples. Thus, determining the serum, tissue, or cerebrospinal
fluid cytokine profile can be used to determine the proper treatment
protocol. For example, two patients having similar PsA symptoms may have
different levels of IL-10 within their serum. The patient with low levels
may benefit from a treatment of IL-10 while the patient with high levels
of IL-10 may benefit from treatment with IL-10 inhibitors such as
anti-IL-10 antibodies. Thus, determining the cytokine profile from a
patient can help provide clinicians and patients with information that
can be used to determine adequate treatments.

[0363] Another embodiment of the invention features a method for
determining the predisposition of a psoriatic arthritis patient to
develop severe disease. The method includes determining the level of a
cytokine (e.g., GM-CSF, IL-17, IL-2, IL-10, IL-13, IFN-γ, IL-6,
CCL4/MIP-1β, and CCL2/MCP-1) within a sample from the patient,
comparing the level of the cytokine to a reference level to obtain
information about the psoriatic arthritis condition. The sample can be
from peripheral blood serum, tissue biopsy (e.g., a synovial tissue
biopsy), cerebrospinal fluid, or other body fluid. The reference level
can be the median level of the cytokine found in samples derived from a
population. The population can include a population of patients having a
mild psoriatic arthritis condition, a population of patients having a
intermediate psoriatic arthritis condition, a population of patients
having a severe psoriatic arthritis condition, a population of healthy
individuals, a population of patients having extra-articular involvement,
or a population of patients having major joint destruction.

[0366] If the cytokine profiles indicate that a patient has severe
disease, then that patient also can be classified as being predisposed to
develop a severe ReA condition including major organ involvement and
major joint destruction. If the cytokine profile indicates that a patient
has mild or intermediate disease, then further analysis can be performed
to determine that patient's predisposition to develop severe disease.

[0367] Specifically, the invention involves analyzing peripheral blood,
serum, plasma, cerebrospinal fluid, a tissue sample, or other body fluid
sample for particular cytokines such as, IL-1α, IL-1β, IL-2,
IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-15, IL-17,
IL-18, INF-α, INF-β, INF-γ, TNF-α, GM-CSF, G-CSF,
MIP-1α/CCL3, MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5,
Eotaxin/CCL11, VEGF, EGF, or FGF. The reference level can be the median
level of the cytokine found in samples derived from a population. The
population can include a population of patients having a mild ReA
condition, a population of patients having a intermediate ReA condition,
a population of patients having a severe ReA condition, a population of
healthy individuals, a population of patients having extra-articular
involvement, or a population of patients having major joint destruction.

[0368] A particular level of a particular cytokine can be determined to be
high or low based on the levels measured, from various populations. Such
populations can include, without limitation, populations of patents with
a mild condition, intermediate condition, or severe condition, patients
with extra-articular involvement, patients with major joint destruction,
and healthy individuals.

[0369] The invention also is based on the discovery that patients
presenting similar ReA symptoms can have different levels of particular
cytokines within their serum, tissue, cerebrospinal fluid, or other body
fluid samples. Thus, determining the serum, tissue, or cerebrospinal
fluid cytokine profile can be used to determine the proper treatment
protocol. For example, two patients having similar ReA symptoms may have
different levels of IL-10 within their serum. The patient with low levels
may benefit from a treatment of IL-10 while the patient with high levels
of IL-10 may benefit from treatment with IL-10 inhibitors such as
anti-IL-10 antibodies. Thus, determining the cytokine profile from a
patient can help provide clinicians and patients with information that
can be used to determine adequate treatments.

[0370] Another embodiment of the invention features a method for
determining the predisposition of a reactive arthritis patient to develop
severe disease. The method includes determining the level of a cytokine
(e.g., IL-12, IFN-γ, IL-13, IL-17, CCL2/MCP-1, TNF-α, IL-4,
G-CSF, and IL-6.) within a sample from the patient, comparing the level
of the cytokine to a reference level to obtain information about the
reactive arthritis condition. The sample can be from peripheral blood,
serum, plasma, tissue biopsy (e.g., a synovial tissue biopsy),
cerebrospinal fluid, or other body fluid. The reference level can be the
median level of the cytokine found in samples derived from a population.
The population can include a population of patients having a mild
reactive arthritis condition, a population of patients having a
intermediate reactive arthritis condition, a population of patients
having a severe reactive arthritis condition, a population of healthy
individuals, a population of patients having extra-articular involvement,
or a population of patients having major joint destruction.

[0373] If the cytokine profiles indicate that a patient has severe
disease, then that patient also can be classified as being predisposed to
develop a severe EA condition including major organ involvement and major
joint destruction. If the cytokine profile indicates that a patient has
mild or intermediate disease, then further analysis can be performed to
determine that patient's predisposition to develop severe disease.

[0374] Specifically, the invention involves analyzing peripheral blood,
serum, plasma, cerebrospinal fluid, a tissue sample, or other body fluid
sample for particular cytokines such as, IL-1α, IL-1β, IL-2,
IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-15, IL-17,
IL-18, INF-α, INF-β, INF-γ, TNF-α, GM-CSF, G-CSF,
MIP-1β/CCL3, MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5,
Eotaxin/CCL11, VEGF, EGF, or FGF. The reference level can be the median
level of the cytokine found in samples derived from a population. The
population can include a population of patients having a mild EA
condition, a population of patients having a intermediate EA condition, a
population of patients having a severe. EA condition, a population of
healthy individuals, a population of patients having extra-articular
involvement, or a population of patients having major joint destruction.

[0375] A particular level of a particular cytokine can be determined to be
high or low based on the levels measured from various populations. Such
populations can include, without limitation, populations of patents with
a mild condition, intermediate condition, or severe condition, patients
with extra-articular involvement, patients with major joint destruction,
and healthy individuals.

[0376] The invention also is based on the discovery that patients
presenting similar EA symptoms can have different levels of particular
cytokines within their serum, tissue, cerebrospinal fluid, or other body
fluid samples. Thus, determining the serum, tissue, or cerebrospinal
fluid cytokine profile can be used to determine the proper treatment
protocol. For example, two patients having similar EA symptoms may have
different levels of IL-10 within their serum. The patient with low levels
may benefit from a treatment of IL-10 while the patient with high levels
of IL-10 may benefit from treatment with IL-10 inhibitors such as
anti-IL-10 antibodies. Thus, determining the cytokine profile from a
patient can help provide clinicians and patients with information that
can be used to determine adequate treatments.

[0377] Another embodiment of the invention features a method for
determining the predisposition of a enteropathic arthritis patient to
develop severe disease. The method includes determining the level of a
cytokine (e.g., CXCL8/IL-8, IL-1β, IL-4, G-CSF, IFN-γ, and
TNF-α.) within a sample from the patient, comparing the level of
the cytokine to a reference level to obtain information about the
enteropathic arthritis condition. The reference level can be the median
level of the cytokine found in samples derived from a population. The
population can include a population of patients having a mild
enteropathic arthritis condition, a population of patients having an
intermediate enteropathic arthritis condition, a population of patients
having a severe enteropathic arthritis condition, a population of healthy
individuals, a population of patients having extra-articular involvement,
or a population of patients having major joint destruction.

[0380] If the cytokine profiles indicate that a patient has severe
disease, then that patient also can be classified as being predisposed to
develop a severe UC condition including major organ involvement and major
joint destruction. If the cytokine profile indicates that a patient has
mild or intermediate disease, then further analysis can be performed to
determine that patient's predisposition to develop severe disease.

[0381] Specifically, the invention involves analyzing peripheral blood,
serum, plasma, cerebrospinal fluid, a tissue sample, or other body fluid
sample for particular cytokines such as, IL-1α, IL-1β, IL-2,
IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-15, IL-17,
IL-18, INF-α, INF-β, INF-γ, TNF-α, GM-CSF, G-CSF,
MIP-1α/CCL3, MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5,
Eotaxin/CCL11, VEGF, EGF, or FGF. The reference level can be the median
level of the cytokine found in samples derived from a population. The
population can include a population of patients having a mild UC
condition, a population of patients having a intermediate UC condition, a
population of patients having a severe UC condition, or a population of
healthy individuals.

[0382] A particular level of a particular cytokine can be determined to be
high or low based on the levels measured from various populations. Such
populations can include, without limitation, populations of patents with
a mild condition, intermediate condition, or severe condition, and
healthy individuals.

[0383] The invention also is based on the discovery that patients
presenting similar UC symptoms can have different levels of particular
cytokines within their peripheral blood, serum, plasma, tissue,
cerebrospinal fluid, or other body fluid samples. Thus, determining the
serum, tissue, or cerebrospinal fluid cytokine profile can be used to
determine the proper treatment protocol. For example, two patients having
similar UC symptoms may have different levels of IL-10 within their
serum. The patient with low levels may benefit from a treatment of IL-10.
Thus, determining the cytokine profile from a patient can help provide
clinicians and patients with information that can be used to determine
adequate treatments.

[0384] Another embodiment of the invention features a method for
determining the predisposition of a ulcerative colitis patient to develop
severe disease. The method includes determining the level of a cytokine
(e.g., IL-7, CXCL8/IL-8, IFN-γ, TNF-α, and IL-10) within a
sample from the patient, comparing the level of the cytokine to a
reference level to obtain information about the psoriatic arthritis
condition and determining if the patient is predisposed to develop severe
disease based on the information. The sample can be from peripheral
blood, serum, plasma, tissue biopsy (e.g., a colon tissue biopsy),
cerebrospinal fluid, or other body fluid. The reference level can be the
median level of the cytokine found in samples derived from a population.
The population can include a population of patients having mild
ulcerative colitis, a population of patients having an intermediate
ulcerative colitis, a population of patients having severe ulcerative
colitis, or a population of healthy individuals.

[0387] If the cytokine profiles indicate that a patient has severe
disease, then that patient also can be classified as being predisposed to
develop a severe CD condition placing the patient at a high risk for
colorectal cancer. If the cytokine profile indicates that a patient has
mild or intermediate disease, then further analysis can be performed to
determine that patient's predisposition to develop severe disease.

[0388] Specifically, the invention involves analyzing peripheral blood,
serum, plasma, cerebrospinal fluid, a tissue sample, or other body fluid
sample for particular cytokines such as, IL-1α, IL-1β, IL-2,
IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-15, IL-17,
IL-18, INF-α, INF-β, INF-γ, TNF-α, GM-CSF, G-CSF,
MIP-1α/CCL3, MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5,
Eotaxin/CCL11, VEGF, EGF, or FGF. The reference level can be the median
level of the cytokine found in samples derived from a population. The
population can include a population of patients having a mild CD
condition, a population of patients having a intermediate CD condition, a
population of patients having a severe CD condition, or a population of
healthy individuals.

[0389] A particular level of a particular cytokine can be determined to be
high or low based on the levels measured from various populations. Such
populations can include, without limitation, populations of patents with
a mild condition, intermediate condition, or severe condition, and
healthy individuals.

[0390] The invention also is based on the discovery that patients
presenting similar CD symptoms can have different levels of particular
cytokines within their serum, tissue, cerebrospinal fluid, or other body
fluid samples. Thus, determining the serum, tissue, or cerebrospinal
fluid cytokine profile can be used to determine the proper treatment
protocol. For example, two patients having similar CD symptoms may have
different levels of IL-10 within their serum or tissue. The patient with
low levels may benefit from a treatment of IL-10. Thus, determining the
cytokine profile from a patient can help provide clinicians and patients
with information that can be used to determine adequate treatments.

[0391] Another embodiment of the invention features a method for
determining the predisposition of a Crohn's disease patient to develop
severe disease. The method includes determining the level of a cytokine
(e.g., TNF-α, IFN-γ, IL-6, IL-7, IL-13, IL-2, IL-4, GM-CSF,
G-CSF, CCL2/MCP-1, and CXCL8/IL-8) within a sample from the patient,
comparing the level of the cytokine to a reference level to obtain
information about the severity of Crohn's disease in the patient. The
sample can be from peripheral blood, serum, plasma, tissue biopsy (e.g.,
colon tissue biopsy), or other body fluid. The reference level can be the
median level of the cytokine found in samples derived from a population.
The population can include a population of patients having mild Crohn's
disease, a population of patients having intermediate Crohn's disease, a
population of patients having a severe Crohn's disease, or a population
of healthy individuals.

[0394] If the cytokine profiles indicate that a patient has severe
disease, then that patient also can be classified as being predisposed to
develop a severe RA condition including major organ involvement and major
joint destruction. If the cytokine profile indicates that a patient has
mild or intermediate disease, then further analysis can be performed to
determine that patient's predisposition to develop severe disease.

[0395] Specifically, the invention involves analyzing peripheral blood,
serum, plasma, cerebrospinal fluid, a tissue sample, or other body fluid
sample for particular cytokines such as, IL-1α, IL-1β, IL-2,
IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-15, IL-17,
IL-18, NF-α, INF-β, INF-γ, TNF-α, GM-CSF, G-CSF,
MIP-1α/CCL3, MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5,
Eotaxin/CCL11, VEGF, EGF, or FGF. The reference level can be the median
level of the cytokine found in samples derived from a population. The
population can include a population of patients having a mild RA
condition, a population of patients having a intermediate RA condition, a
population of patients having a severe RA condition, a population of
healthy individuals, a population of patients having extra-articular
involvement, or a population of patients having major joint destruction.

[0396] A particular level of a particular cytokine can be determined to be
high or low based on the levels measured from various populations. Such
populations can include, without limitation, populations of patents with
a mild condition, intermediate condition, or severe condition, patients
with extra-articular involvement, patients with major joint destruction,
and healthy individuals.

[0397] The invention also is based on the discovery that patients
presenting similar RA symptoms can have different levels of particular
cytokines within their peripheral blood, serum, plasma, tissue,
cerebrospinal fluid, or other body fluid samples. Thus, determining the
serum, tissue, or cerebrospinal fluid cytokine profile can be used to
determine the proper treatment protocol. For example, two patients having
similar RA symptoms may have different levels of IL-10 within their
serum. The patient with low levels may benefit from a treatment of IL-10
while the patient with high levels of IL-10 may benefit from treatment
with IL-10 inhibitors such as anti-IL-10 antibodies. Thus, determining
the cytokine profile from a patient can help provide clinicians and
patients with information that can be used to determine adequate
treatments.

[0398] Another embodiment of the invention features a method for
determining the predisposition of a rheumatoid arthritis patient to
develop severe disease. The method includes determining the level of a
cytokine (e.g., IFN-γ, IL-1β, TNF-α, G-CSF, GM-CSF,
IL-6, IL-4, IL-10, IL-13, IL-5, and IL-7) within a sample from the
patient, comparing the level of the cytokine to a reference level to
obtain information about the rheumatoid arthritis condition. The sample
can be from peripheral blood, serum, plasma, tissue biopsy (e.g., a
synovial tissue biopsy), cerebrospinal fluid, or other body fluid. The
reference level can be the median level of the cytokine found in samples
derived from a population. The population can include a population of
patients having a mild rheumatoid arthritis condition, a population of
patients having a intermediate rheumatoid arthritis condition, a
population of patients having a severe rheumatoid arthritis condition, a
population of healthy individuals, a population of patients having
extra-articular involvement, or a population of patients having major
joint destruction.

[0401] If the cytokine profiles indicate that a patient has severe
disease, then that patient also can be classified as being predisposed to
develop a severe SLE condition including major organ involvement and
major joint destruction. If the cytokine profile indicates that a patient
has mild or intermediate disease, then further analysis can be performed
to determine that patient's predisposition to develop severe disease.

[0402] Specifically, the invention involves analyzing peripheral blood,
serum, plasma, cerebrospinal fluid, a tissue sample, or other body fluid
sample for particular cytokines such as, IL-1α, IL-1β, IL-2,
IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-15, IL-17,
IL-18, INF-α, INF-β, INF-γ, TNF-α, GM-CSF, G-CSF,
MIP-1α/CCL3, MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5,
Eotaxin/CCL11, VEGF, EGF, or FGF. The reference level can be the median
level of the cytokine found in samples derived from a population. The
population can include a population of patients having a mild SLE
condition, a population of patients having a intermediate SLE condition,
a population of patients having a severe SLE condition, a population of
healthy individuals, a population of patients having extra-articular
involvement, or a population of patients having major joint destruction.

[0403] A particular level of a particular cytokine can be determined to be
high or low based on the levels measured from various populations. Such
populations can include, without limitation, populations of patents with
a mild condition, intermediate condition, or severe condition, patients
with extra-articular involvement, patients with major joint destruction,
and healthy individuals.

[0404] The invention also is based on the discovery that patients
presenting similar SLE symptoms can have different levels of particular
cytokines within their serum, tissue, cerebrospinal fluid, or other body
fluid samples. Thus, determining the serum, tissue, or cerebrospinal
fluid cytokine profile can be used to determine the proper treatment
protocol. For example, two patients having similar SLE symptoms may have
different levels of IL-10 within their serum. The patient with low levels
may benefit from a treatment of IL-10 while the patient with high levels
of IL-10 may benefit from treatment with IL-10 inhibitors such as
anti-IL-10 antibodies. Thus, determining the cytokine profile from a
patient can help provide clinicians and patients with information that
can be used to determine adequate treatments.

[0405] Another embodiment of the invention features a method for
determining the predisposition of the systemic lupus erythematosus
patient to develop severe disease. The method includes determining the
level of a cytokine (e.g., IL-10, IL-2, IL-4, IL-6, IFN-γ,
CCL2/MCP-1, CXCL8/IL-8, and IL-17.) within a sample from the patient,
comparing the level of the cytokine to a reference level to obtain
information about the SLE condition. The sample can be from peripheral
blood, serum, plasma, tissue biopsy (e.g., a synovial tissue biopsy),
cerebrospinal fluid, or other body fluid. The reference level can be the
median level of the cytokine found in samples derived from a population.
The population can include a population of patients having a mild
systemic lupus erythematosus condition, a population of patients having
an intermediate systemic lupus erythematosus condition, a population of
patients having a severe systemic lupus erythematosus condition, a
population of healthy individuals, a population of patients having
extra-articular involvement, or a population of patients having major
joint destruction.

[0408] If the cytokine profiles indicate that a patient has severe
disease, then that patient also can be classified as being predisposed to
develop a severe FMF condition including major organ involvement and
major joint destruction. If the cytokine profile indicates that a patient
has mild or intermediate disease, then further analysis can be performed
to determine that patient's predisposition to develop severe disease.

[0409] Specifically, the invention involves analyzing peripheral blood
serum, cerebrospinal fluid, a tissue sample, or other body fluid sample
for particular cytokines such as, IL-1α, IL-1β, IL-2, IL-4,
IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18,
INF-α, INF-β, INF-γ, TNF-α, GM-CSF, G-CSF,
MIP-1α/CCL3, MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5,
Eotaxin/CCL11, VEGF, EGF, or FGF The reference level can be the median
level of the cytokine found in samples derived from a population. The
population can include a population of patients having a mild FMF
condition, a population of patients having a intermediate FMF condition,
a population of patients having a severe FMF condition, a population of
healthy individuals, a population of patients having extra-articular
involvement, or a population of patients having major joint destruction.

[0410] A particular level of a particular cytokine can be determined to be
high or low based on the levels measured from various populations. Such
populations can include, without limitation, populations of patents with
a mild condition, intermediate condition, or severe condition, patients
with extra-articular involvement, patients with major joint destruction,
and healthy individuals.

[0411] The invention also is based on the discovery that patients
presenting similar FMF symptoms can have different levels of particular
cytokines within their serum, tissue, cerebrospinal fluid, or other body
fluid samples. Thus, determining the serum, tissue, or cerebrospinal
fluid cytokine profile can be used to determine the proper treatment
protocol. For example, two patients having similar FMF symptoms may have
different levels of IL-8 within their serum. A patient with high levels
of IL-8 may benefit from treatment with IL-8 antagonists. Thus,
determining the cytokine profile from a patient can help provide
clinicians and patients with information that can be used to determine
adequate treatments.

[0412] Another embodiment of the invention features a method for
determining the predisposition of a familial Mediterranean fever patient
to develop severe disease. The method includes determining the level of a
cytokine (e.g., IL-17, IL-6, CCL2/MCP-1, TNF-α INF-γ, GM-CSF,
IL-13, IL-4, G-CSF, and CXCL8/IL-8) within a sample from the patient,
comparing the level of the cytokine to a reference level to obtain
information about the familial Mediterranean fever condition. The sample
can be from peripheral blood, serum, plasma, tissue biopsy (e.g., a
synovial tissue biopsy), cerebrospinal fluid, or other body fluid. The
reference level can be the median level of the cytokine found in samples
derived from a population. The population can include a population of
patients having a mild familial Mediterranean fever condition, a
population of patients having an intermediate familial Mediterranean
fever condition, a population of patients having a severe familial
Mediterranean fever condition, a population of healthy individuals, a
population of patients having extra-articular involvement, or a
population of patients having major joint destruction.

[0415] Specifically, the invention involves analyzing cerebrospinal fluid,
other body fluid or tissue samples for particular cytokines such as,
IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10,
IL-12, IL-13, IL-15, IL-17, IL-18, INF-α, INF-β, INF-γ,
TNF-α, GM-CSF, G-CSF, MIP-1α/CCL3, MIP-1β/CCL4,
MCP-1/CCL2, RANTES/CCL5, Eotaxin/CCL11, VEGF, EGF, or FGF. The reference
level can be the median level of the cytokine found in samples derived
from a population. A particular level of a particular cytokine can be
determined to be high or low based on the levels measured from various
populations.

[0416] The invention also is based on the discovery that patients
presenting similar ALS symptoms can have different levels of particular
cytokines within their cerebrospinal fluid, other body fluid, or tissue
samples. Thus, determining the cerebrospinal fluid, body fluid, or tissue
samples cytokine profile can be used to determine the proper treatment
protocol. For example, two patients having similar ALS symptoms may have
different levels of IL-12 within their serum. A patient with low levels
of IL-12 may benefit from treatment with IL-12 agonists. Thus,
determining the cytokine profile from a patient can help provide
clinicians and patients with information that can be used to determine
adequate treatments.

[0419] If the cytokine profiles indicate that a patient has severe
disease, then that patient also can be classified as being predisposed to
develop a severe gastrointestinal condition including major organ
involvement with an increased risk in gastrointestinal cancers.

[0420] Specifically, the invention involves analyzing peripheral blood,
serum, plasma, a tissue sample, or other body fluid sample for particular
cytokines such as, IL-1α, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8,
IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, INF-α, INF-β,
INF-γ, TNF-α, GM-CSF, G-CSF, MIP-1α/CCL3,
MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5, Eotaxin/CCL11, VEGF, EGF, or
FGF. The reference level can be the median level of the cytokine found in
samples derived from a population. The population can include a
population of patients having a mild IBS condition, a population of
patients having a intermediate IBS condition, a population of patients
having a severe IBS condition, or a population of healthy individuals.

[0421] A particular level of a particular cytokine can be determined to be
high or low based on the levels measured from various populations. Such
populations can include, without limitation, populations of patents with
a mild condition, intermediate condition, or severe condition, and
patients with extra-gatrointestinal immune system involvement.

[0422] The invention is also based on the discovery that patients
presenting similar IBS symptoms can have different levels of particular
cytokines within their serum, tissue, or other body fluid samples. Thus,
determining the serum, tissue, or other fluid cytokine profile can be
used to determine the proper treatment protocol. For example, two
patients having similar IBS symptoms may have different levels of
CXCL8/IL-8 within their serum. The patient with high levels may benefit
from a treatment of an anti-CXCL8/IL-8 drug or biologic while the
patients with low levels of CXCL8/IL-8 may benefit from less aggressive
treatment alternatives. Thus, determining the cytokine profile from a
patient can help provide clinicians and patients with information that
can be used to determine adequate treatments.

[0423] Another embodiment of the invention features a method for
determining the predisposition of the IBS patient to develop severe
disease. The method includes determining the level of a cytokine (e.g.,
TNF-α, IFN-γ, IL-1β, IL-6, IL-7, GM-CSF, G-CSF,
CCL2/MCP-1, and CXCL8/IL-8) within a sample from the patient, comparing
the level of the cytokine to a reference level to obtain information
about the IBS condition. The sample can be from peripheral blood, serum,
plasma, tissue biopsy (e.g., colonic tissue biopsy), or other body fluid.
The reference level can be the median level of the cytokine found in
samples derived from a population. The population can include a
population of patients having a mild IBS condition, a population of
patients having an intermediate IBS condition, a population of patients
having a severe IBS condition, or a population of healthy individuals.

[0426] If the cytokine profiles indicate that a patient has severe
disease, then that patient also can be classified as being predisposed to
develop a severe JRA condition including major organ involvement and
major joint destruction. If the cytokine profile indicates that a patient
has mild or intermediate disease, then further analysis can be performed
to determine that patient's predisposition to develop severe disease.

[0427] Specifically, the invention involves analyzing peripheral blood,
serum, plasma, cerebrospinal fluid, a tissue sample, or other body fluid
sample for particular cytokines such as, IL-1α, IL-1β, IL-2,
IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-15, IL-17,
IL-18, INF-α, INF-β, INF-γ, TNF-α, GM-CSF, G-CSF,
MIP-1α/CCL3, MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5,
Eotaxin/CCL11, VEGF, EGF, or FGF. The reference level can be the median
level of the cytokine found in samples derived from a population. The
population can include a population of patients having a mild JRA
condition, a population of patients having a intermediate JRA condition,
a population of patients having a severe JRA condition, a population of
healthy individuals, a population of patients having extra-articular
involvement, or a population of patients having major joint destruction.

[0428] A particular level of a particular cytokine can be determined to be
high or low based on the levels measured from various populations. Such
populations can include, without limitation, populations of patents with
a mild condition, intermediate condition, or severe condition, patients
with extra-articular involvement, patients with major joint destruction,
and healthy individuals.

[0429] The invention is also based on the discovery that patients
presenting similar JRA symptoms can have different levels of particular
cytokines within their serum, tissue, cerebrospinal fluid, or other body
fluid samples. Thus, determining the serum, tissue, or cerebrospinal
fluid cytokine profile can be used to determine the proper treatment
protocol. For example, two patients having similar JRA symptoms may have
different levels of IL-1β within their serum. The patient with high
levels of IL-1β may benefit from treatment with IL-1β
inhibitors. Thus, determining the cytokine profile from a patient can
help provide clinicians and patients with information that can be used to
determine adequate treatments.

[0430] Another embodiment of the invention features a method for
determining the predisposition of a JRA patient to develop severe
disease. The method includes determining the level of a cytokine (e.g.,
IFN-γ, IL-1β, TNF-α, G-CSF, GM-CSF, IL-6, IL-4, IL-10,
IL-13, IL-5, and IL-7) within a sample from the patient, comparing the
level of the cytokine to a reference level to obtain information about
the JRA condition. The sample can be from peripheral blood, serum,
plasma, tissue biopsy (e.g., a synovial tissue biopsy), cerebrospinal
fluid, or other body fluid. The reference level can be the median level
of the cytokine found in samples derived from a population. The
population can include a population of patients having a JRA condition, a
population of patients having a intermediate JRA condition, a population
of patients having a severe JRA condition, a population of healthy
individuals, a population of patients having extra-articular involvement,
or a population of patients having major joint destruction.

[0433] Specifically, the invention involves analyzing peripheral blood,
serum, plasma, a tissue sample, or other body fluid sample for particular
cytokines such as, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7,
IL-8/CXCL8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, INF-α,
INF-β, INF-γ, TNF-α, GM-CSF, G-CSF, MIP-1α/CCL3,
MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5, Eotaxin/CCL11, VEGF, EGF, or
FGF. The reference level can be the median level of the cytokine found in
samples derived from a population. The population can include a
population of patients having a mild SS condition, a population of
patients having a intermediate SS condition, a population of patients
having a severe SS condition, a population of healthy individuals, a
population of patients having extra-articular involvement, or a
population of patients having major joint destruction.

[0434] A particular level of a particular cytokine can be determined to be
high or low based on the levels measured from various populations. Such
populations can include, without limitation, populations of patents with
a mild condition, intermediate condition, or severe condition, patients
with extra-articular involvement, patients with major joint destruction,
and healthy individuals.

[0435] The invention is also ibased on the discovery that patients
presenting similar SS symptoms can have different levels of particular
cytokines within their serum, tissue, cerebrospinal fluid, or other body
fluid samples. Thus, determining the serum, tissue, or cerebrospinal
fluid cytokine profile can be used to determine the proper treatment
protocol. For example, two patients having similar SS symptoms may have
different levels of IL-2 within their serum. patient with high levels of
IL-2 may benefit from treatment with IL-2 inhibitors such as anti-IL-2
biological drugs. Thus, determining the cytokine profile from a patient
can help provide clinicians and patients with information that can be
used to determine adequate treatments.

[0436] Another embodiment of the invention features a method for
determining the predisposition of the SS patient to develop severe
disease. The method includes determining the level of a cytokine (e.g.,
CCL2/MCP-1, IL-12, CXCL8/IL-8, CCL11/Eotaxin, TNFα, IL-2,
IFNα, IL-15, IL17, IL-1α, IL-1β, IL-6, and GM-CSF)
within a sample from the patient, comparing the level of the cytokine to
a reference level to obtain information about the SS condition. The
sample can be from peripheral blood, serum, plasma, tissue biopsy (e.g.,
a salivary gland or lip tissue biopsy), or other body fluid. The
reference level can be the median level of the cytokine found in samples
derived from a population.

[0439] If the cytokine profiles indicate that a patient has severe
disease, then that patient also can be classified as being predisposed to
develop a severe EArth condition including major organ involvement and
major joint destruction. If the cytokine profile indicates that a patient
has mild or intermediate disease, then further analysis can be performed
to determine that patient's predisposition to develop severe disease.

[0440] Specifically, the invention involves analyzing peripheral blood,
serum, plasma, cerebrospinal fluid, a tissue sample, or other body fluid
sample for particular cytokines such as, IL-1α, IL-1β, IL-2,
IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-15, IL-17,
IL-18, INF-α, INF-β, INF-γ, TNF-α, GM-CSF, G-CSF,
MIP-1α/CCL3, MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5,
Eotaxin/CCL11, VEGF, EGF, or FGF. The reference level can be the median
level of the cytokine found in samples derived from a population. The
population can include a population of patients having a mild EArth
condition, a population of patients having a intermediate EArth
condition, a population of patients having a severe EArth condition, a
population of healthy individuals, a population of patients having
extra-articular involvement, or a population of patients having major
joint destruction.

[0441] A particular level of a particular cytokine can be determined to be
high or low based on the levels measured from various populations. Such
populations can include, without limitation, populations of patents with
a mild condition, intermediate condition, or severe condition, patients
with extra-articular involvement, patients with major joint destruction,
and healthy individuals.

[0442] The invention is also based on the discovery that patients
presenting similar EArth symptoms can have different levels of particular
cytokines within their serum, tissue, cerebrospinal fluid, or other body
fluid samples. Thus, determining the serum, tissue, or cerebrospinal
fluid cytokine profile can be used to determine the proper treatment
protocol. For example, two patients having similar EArth symptoms may
have different levels of IL-6 within their serum. The patient with high
levels of IL-6 may benefit from treatment with IL-6 inhibitors. Thus,
determining the cytokine profile from a patient can help provide
clinicians and patients with information that can be used to determine
adequate treatments.

[0443] Another embodiment of the invention features a method for
determining the predisposition of the EArth patient to develop severe
disease. The method includes determining the level of a cytokine (e.g.,
IL-6. IL-2, IL-12, GM-CSF, IL-5, IL-4, CCL4/MIP-1β, and CXCL8/IL-8)
within a sample from the patient, comparing the level of the cytokine to
a reference level to obtain information about the psoriatic arthritis
condition. The sample can be from peripheral blood, serum, plasma, tissue
biopsy (e.g., a synovial tissue biopsy), cerebrospinal fluid, or other
body fluid. The reference level can be the median level of the cytokine
found in samples derived from a population.

[0446] If the cytokine profiles indicate that a patient has severe
disease, then that patient also can be classified as being predisposed to
develop a severe PSO condition including major organ involvement and
major joint destruction. If the cytokine profile indicates that a patient
has mild or intermediate disease, then further analysis can be performed
to determine that patient's predisposition to develop severe disease.

[0447] Specifically, the invention involves analyzing peripheral blood,
serum, plasma, cerebrospinal fluid, a tissue sample, or other body fluid
sample for particular cytokines such as, IL-1α, IL-1β, IL-2,
IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-15, IL-17,
IL-18, INF-α, INF-β, INF-γ, TNF-α, GM-CSF, G-CSF,
MIP-1α/CCL3, MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5,
Eotaxin/CCL11, VEGF, EGF, or FGF. The reference level can be the median
level of the cytokine found in samples derived from a population. The
population can include a population of patients having a mild PSO
condition, a population of patients having a intermediate PSO condition,
a population of patients having a severe PSO condition, a population of
healthy individuals, a population of patients having extra-articular
involvement, or a population of patients having major joint destruction.

[0448] A particular level of a particular cytokine can be determined to be
high or low based on the levels measured from various populations. Such
populations can include, without limitation, populations of patents with
a mild condition, intermediate condition, or severe condition.

[0449] The invention is also based on the discovery that patients
presenting similar PSO symptoms can have different levels of particular
cytokines within their skin, serum, tissue, or other body fluid samples.
Thus, determining the skin, serum, tissue, or cytokine profile can be
used to determine the proper treatment protocol. For example, two
patients having similar PSO symptoms may have different levels of IL-6
within their serum. The patient with high levels of IL-6 may benefit from
treatment with IL-6 inhibitors. Thus, determining the cytokine profile
from a patient can help provide clinicians and patients with information
that can be used to determine adequate treatments.

[0450] Another embodiment of the invention features a method for
determining the predisposition of the PSO patient to develop severe
disease. The method includes determining the level of a cytokine (e.g.,
IL-6, IL-10, IL-2, IL-4, IFN-γ, CCL2/MCP-1, and IL-17.) within a
sample from the patient, comparing the level of the cytokine to a
reference level to obtain information about the PSO condition. The sample
can be from peripheral blood, serum, plasma, skin, tissue biopsy, or
other body fluid. The reference level can be the median level of the
cytokine found in samples derived from a population.

[0453] If the cytokine profiles indicate that a patient has severe
disease, then that patient also can be classified as being predisposed to
develop a severe NI condition including central nervous system
involvement and peripheral nervous system involvement. If the cytokine
profile indicates that a patient has mild or intermediate disease, then
further analysis can be performed to determine that patient's
predisposition to develop severe disease.

[0454] Specifically, the invention involves analyzing peripheral blood,
serum, plasma, cerebrospinal fluid, a tissue sample, or other body fluid
sample for particular cytokines such as, IL-1α, IL-1β, IL-2,
IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-15, IL-17,
IL-18, INF-α, INF-β, INF-γ, TNF-α, GM-CSF, G-CSF,
MIP-1α/CCL3, MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5,
Eotaxin/CCL11, VEGF, EGF, or FGF. The reference level can be the median
level of the cytokine found in samples derived from a population. The
population can include a population of patients having a mild NI
condition, a population of patients having a intermediate NI condition, a
population of patients having a severe NI condition, a population of
healthy individuals, a population of patients having a central nervous
system condition, or a population of patients having a peripheral nervous
system condition.

[0455] A particular level of a particular cytokine can be determined to be
high or low based on the levels measured from various populations. Such
populations can include, without limitation, populations of patents with
a mild condition, intermediate condition, or severe condition, a
population of patients having a central nervous system condition, or a
population of patients having a peripheral nervous system condition.

[0456] The invention is also based on the discovery that patients
presenting similar NI symptoms can have different levels of particular
cytokines within their serum, tissue, cerebrospinal fluid, or other body
fluid samples. Thus, determining the serum, tissue, or cerebrospinal
fluid cytokine profile can be used to determine the proper treatment
protocol. For example, two patients having similar NI symptoms may have
different levels of CCL2/MCP-1 within their cerebrospinal fluid. The
patient with high levels of CCL2/MCP-1 may benefit from treatment with
CCL2/MCP-1inhibitors. Thus, determining the cytokine profile from a
patient can help provide clinicians and patients with information that
can be used to determine adequate treatments.

[0457] Another embodiment of the invention features a method for
determining the predisposition of the NI patient to develop severe
disease. The method includes determining the level of a cytokine (e.g.,
CCL2/MCP-1, IL-12, GM-CSF, G-CSF, M-CSF, IL-6, and IL-17.) within a
sample from the patient, comparing the level of the cytokine to a
reference level to obtain information about the NI condition. The sample
can be from peripheral blood, serum, plasma, tissue biopsy (e.g., a
synovial tissue biopsy), cerebrospinal fluid, or other body fluid. The
reference level can be the median level of the cytokine found in samples
derived from a population.

V. EXAMPLES

[0458] Specific embodiments of the invention will now be further described
by the following, nonlimiting examples which will serve to illustrate in
some detail various features. The following examples are included to
facilitate an understanding of ways in which the invention may be
practiced. It should be appreciated that the examples which follow
represent embodiments discovered to function well in the practice of the
invention, and thus can be considered to constitute preferred modes for
the practice of the invention. However, it should be appreciated that
many changes can be made in the exemplary embodiments which are disclosed
while still obtaining like or similar result without departing from the
spirit and scope of the invention. Accordingly, the examples should not
be construed as limiting the scope of the invention.

Example 1

Distinct Cytokine Patterns in Ankylosing Spondylitis (AS)

[0459] A distinct profile of cytokines was generated from patients with
ankylosing spondylitis (AS). This cytokine profile was determined by
sampling peripheral blood serum for the presence of cytokines. Patients
were found to have predictive molecular cytokine profiles based on
clinical disease phenotype and disease severity. Specifically, cytokines
were found to be similar within the specific disease classifications, but
the levels of cytokines were somewhat heterogeneous with regard to
individual patient, raising the possibility that various stages of
disease and disease severity may be distinguished by this molecular
diagnostic mechanism.

[0460] 1. Study Population

[0461] Peripheral blood serum was obtained from more than 44 patients with
active ankylosing spondylitis who fulfilled the diagnostic criteria and
classification by the European Spondylarthropathy Study Group (ESSG)
--Bath Criteria (Calin, A., J. D. Taurog., Eds. 1998, "The
Spondylarthritides.")

[0465] The clinical presentations of the cytokine profiles in the defined
patient subsets were compared by using a standard Student's t-test,
stepwise discriminate function tests, and a robust cluster analysis.
Differentially expressed cytokines were identified first by a paired
Student's t-test with the commonly accepted significance threshold of
p<0.05. The Student's t-test was carried out using Microsoft Excel
(Microsoft, Inc., Redman Wash.). Taking into consideration individual
variances among the patient population, a second method of selection was
also utilized.

[0466] Discriminant function analysis (DFA) was used for selection of the
set of cytokines with maximal discriminatory capabilities between groups
of samples from patients and unaffected controls. We used variant DFA
named the Forward Stepwise Analysis.

[0467] In stepwise DFA, the model for discrimination is built
step-by-step. Specifically, at each step we review all variables and
evaluate which one will contribute most to the discrimination between
groups. This variable is then included in the model, and the process
proceeds to the next step. The stepwise procedure is "guided" by the
respective F to enter values. The F value for a variable indicates its
statistical significance in the discrimination between groups, that is,
it is a measure of the extent to which a variable makes a unique
contribution into the discriminative functions--roots, which are linear
combinations of the gene expressions with constant coefficients and used
for the prediction of group membership. In general, we continue to choose
variables to be included in the model, as long as the respective F values
for those variables are larger than the user-specified F to enter.

[0468] The statistical significance of discriminative power of each
cytokine can also characterized by partial Wilk's Lambda--equivalent to
the partial correlation coefficient in multiple regression analysis.
Wilk's Lambda is a ratio of within group difference to the sum of within
plus between group differences (standard deviations). Its value ranges
from 1.0 (no discriminatory power) to 0.0 (perfect discriminatory power).

[0469] DFA was carried out with use of the package Statistica (StatSoft,
Tulsa Okla.). This software produces list of cytokines having maximal
discriminatory capability and roots--linear combinations of these
cytokines having similar within group values and different for each
group. The discriminant potential of the final equations can be observed
in a simple multi-dimensional plot of the values of the roots obtained
for each group. This provides a graphical representation of the
similarity among the various groups.

[0470] Variant of cluster analysis with the Pearson correlation
coefficient as a measure of similarity and the threshold r=0.8 was used
for further characterization of samples heterogeneity. Data obtained from
AS patients, HLA B27 positive unaffected controls, and HLA B27 negative
unaffected controls were analyzed with these methods.

[0471] 4. Results

[0472] The ankylosing spondylitis patients shared similar cytokine
profiles. MHC Class I HLA-B27 positive healthy controls also shared
distinct cytokine characteristics with patients, but not with HLA-B27
negative controls, suggesting that HLA-B27 plays a principal role in both
the etiology and pathophysiology of spondyloarthritic disease. Ankylosing
spondylitis (AS) has a strong genetic association with the MHC I-HLA-B27
locus. However, the role of HLA-B27 in AS pathophysiology remains
controversial.

[0473] Using a comprehensive multiplex cytokine assay we found a specific
set of cytokines that are upregulated in the peripheral blood of
ankylosing spondylitis patients including: CCL4/MIP-1β, IL-17, IL-6,
CCL2/MCP-1, TNF-α, GM-CSF, IL-13, IL-4, G-CSF, and CXCL8/IL-8.
HLA-B27 positive healthy controls also had elevated IFN-γ (p=0.07)
and IL-8 (p=0.08) levels compared to HLA-B27 negative controls,
suggesting that HLA-B27 may function to predispose patients to disease.
Levels of IL-1β were not significantly elevated in AS patients, even
when levels of TNF-α and IFN-γ were significantly elevated.
The cytokines upregulated in these patients are unique to AS (this group
was not characteristic of other spondyloarthropathy subtypes, rheumatoid
arthritis, or SLE), suggesting this complex immune activity specifically
induces spondylitis and/or sacroilitis. Moreover, cytokine titers
correlated with disease activity, suggesting that this group of cytokines
can be used to guide biologic therapy and as a surrogate of disease
activity in AS.

Example 2

Distinct Cytokine Patterns in Psoriatic Arthritis (PsA)

[0474] A distinct profile of cytokines was geneated from patients with
psoriatic arthritis (PsA). This distinct cytokine profile was determined
by sampling serum and cerebrospinal fluid for the presence of cytokines.
Patients were found to have predictive molecular cytokine profiles based
on clinical disease phenotype and disease severity. Specifically,
cytokines were found to be similar within the specific disease
classifications, but the levels of cytokines were somewhat heterogeneous
with regard to individual patient, raising the possibility that various
stages of disease and disease severity may be distinguished by this
molecular diagnostic mechanism.

[0475] 1. Study Population

[0476] Peripheral blood serum from seventy-two (72) patients diagnosed
with psoriatic arthritis were analyzed. Cerebrospinal fluid from one
patient was also analyzed.

[0480] The clinical presentations of the cytokine profiles in the defined
patient subsets were compared by using a standard Student's t-test,
stepwise discriminate function tests, and a robust cluster analysis.
Differentially expressed cytokines were identified first by a paired
Student's t-test with the commonly accepted significance threshold of
p<0.05. The Student's t-test was carried out using Microsoft Excel
(Microsoft, Inc., Redman Wash.). Taking into consideration individual
variances among the patient population, a second method of selection was
also utilized.

[0481] Discriminant function analysis (DFA) (described above) was used for
selection of the set of cytokines with maximal discriminatory
capabilities between groups of samples from patients and unaffected
controls. The inventors used variant DFA named the Forward Stepwise
Analysis.

[0482] 4. Results

[0483] The data presented herein indicate the contribution of different
cytokines in controlling inflammation and also emphasize the necessity to
re-evaluate the role of the immune pathways in the pathogenesis of
psoriatic arthritis. Immune deviation is now widely accepted as a concept
in explaining the pathogenesis of autoimmune diseases. This model implies
that chronic inflammation is a consequence of the aberrant commitment to
an immune pathway in response to a given antigen (Finkelman F D, J Exp
Med, pp. 182:279-282, 1995).

[0485] A distinct profile of cytokines was produced from patients with
reactive arthritis (ReA). This distinct cytokine profile was determined
by sampling serum for the presence of cytokines. Patients were found to
have predictive molecular cytokine profiles based on clinical disease
phenotype and disease severity. Specifically, cytokines were found to be
similar within the specific disease classifications, but the levels of
cytokines were somewhat heterogeneous with regard to individual patient,
raising the possibility that various stages of disease and disease
severity may be distinguished by this molecular diagnostic mechanism.

[0491] The clinical presentations of the cytokine profiles in the defined
patient subsets were compared by using a standard Student's t-test,
stepwise discriminate function tests, and a robust cluster analysis.
Differentially expressed cytokines were identified first by a paired
Student's t-test with the commonly accepted significance threshold of
p<0.05. The Student's t-test was carried out using Microsoft Excel
(Microsoft, Inc., Redman Wash.). Taking into consideration individual
variances among the patient population, a second method of selection was
also utilized.

[0492] Discriminant function analysis (DFA) (described above) was used for
selection of the set of cytokines with maximal discriminatory
capabilities between groups of samples from patients and unaffected
controls. We used variant DFA named the Forward Stepwise Analysis.

[0493] 4. Results

[0494] The data presented herein indicate the contribution of different
cytokines in controlling inflammation and also emphasize the necessity to
re-evaluate the role of the immune pathways in the pathogenesis of
reactive arthritis. Immune deviation is now widely accepted as a concept
in explaining the pathogenesis of autoimmune diseases (Finkelman F D, J
Exp Med, pp. 182:279-282 (1995).

[0495] Reactive arthritis (ReA) is a chronic systemic inflammatory
rheumatic disorder of e axial skeleton with or without extraskeletal
manifestations. Mechanisms underlying the phenotypic heterogeneity of
reactive arthritis are not known, but bacterial and viral antigens are
suspected to contribute to disease etiology. To explore whether a
molecular diagnostic characteristic exists, serum samples from 13
patients with clinically diagnosed. ReA were examined and the
concentration of cytokines were quantified.

[0497] A distinct profile of cytokines was produced from patients with
enteropathic arthritis (EA). This distinct cytokine profile was
determined by sampling serum for the presence of cytokines. Patients were
found to have predictive molecular cytokine profiles based on clinical
disease phenotype and disease severity. Specifically, cytokines were
found to be similar within the specific disease classifications, but the
levels of cytokines were somewhat heterogeneous with regard to individual
patient, raising the possibility that various stages of disease and
disease severity may be distinguished by this molecular diagnostic
mechanism.

[0503] The clinical presentations of the cytokine profiles in the defined
patient subsets were compared by using a standard Student's t-test,
stepwise discriminate function tests, and a robust cluster analysis.
Differentially expressed cytokines were identified first by a paired
Student's t-test with the commonly accepted significance threshold of
p<0.05. The Student's t-test was carried out using Microsoft Excel
(Microsoft, Inc., Redman Wash.). Taking into consideration individual
variances among the patient population, a second method of selection was
also utilized.

[0504] Discriminant function analysis (DFA) (described above) was used for
selection of the set of cytokines with maximal discriminatory
capabilities between groups of samples from patients and unaffected
controls. We used variant DFA named the Forward Stepwise Analysis.

[0505] 4. Results

[0506] The data presented herein indicate the contribution of different
cytokines in controlling inflammation and also emphasize the necessity to
re-evaluate the role of the immune pathways in the pathogenesis of EA.
Immune deviation is now widely accepted as a concept in explaining the
pathogenesis of autoimmune diseases (FD Finkelman, J Exp Med, pp.
182:279-282, 1995).

[0507] Enteropathic arthritis is a chronic systemic inflammatory rheumatic
disorder of the axial skeleton with or without extraskeletal
manifestations. Mechanisms underlying the phenotypic heterogeneity of
enteropathic arthritis are not known, but gastrointestinal bacterial
antigens are suspected to play an important role in the development of
the disease. To explore whether a molecular diagnostic characteristic
exists, serum samples from 12 patients with clinically diagnosed with
enteropathic arthritis were examined and the concentration of cytokines
were quantified.

[0509] A distinct profile of cytokines was produced from patients with
Ulcerative Colitis (UC). This distinct cytokine profile was determined by
sampling serum for the presence of cytokines. Patients were found to have
predictive molecular cytokine profiles based on clinical disease
phenotype and disease severity. Specifically, cytokines were found to be
similar within the specific disease classifications, but the levels of
cytokines were somewhat heterogeneous with regard to individual patient,
raising the possibility that various stages of disease and disease
severity may be distinguished by this molecular diagnostic mechanism.

[0515] The clinical presentations of the cytokine profiles in the defined
patient subsets were compared by using a standard Student's t-test,
stepwise discriminate function tests, and a robust cluster analysis.
Differentially expressed cytokines were identified first by a paired
Student's t-test with the commonly accepted significance threshold of
p<0.05. The Student's t-test was carried out using Microsoft Excel
(Microsoft, Inc., Redman Wash.). Taking into consideration individual
variances among the patient population, a second method of selection was
also utilized.

[0516] Discriminant function analysis (DFA) (described above) was used for
selection of the set of cytokines with maximal discriminatory
capabilities between groups of samples from patients and unaffected
controls. The inventors used variant DFA named the Forward Stepwise
Analysis.

[0517] 4. Results

[0518] The data presented herein indicate the contribution of different
cytokines in controlling inflammation and also emphasize the necessity to
re-evaluate the role of the immune pathways in the pathogenesis of UC.

[0519] Ulcerative colitis is a chronic systemic inflammatory disorder of
the gastrointestinal system with or without extraintestinal
manifestations. Mechanisms underlying the phenotypic heterogeneity of
irritable bowel disease are not known. To explore whether a molecular
diagnostic characteristic exists, tissue biopsy samples from 10 patients
with clinically diagnosed UC were examined and the concentration of
cytokines were quantified.

[0521] A distinct profile of cytokines was produced from patients with
Crohn's Disease (CD). This distinct cytokine profile was determined by
sampling serum for the presence of cytokines. Patients were found to have
predictive molecular cytokine profiles based on clinical disease
phenotype and disease severity. Specifically, cytokines were found to be
similar within the specific disease classifications, but the levels of
cytokines were somewhat heterogeneous with regard to individual patient,
raising the possibility that various stages of disease and disease
severity may be distinguished by this molecular diagnostic mechanism.

[0527] The clinical presentations of the cytokine profiles in the defined
patient subsets were compared by using a standard Student's t-test,
stepwise discriminate function tests, and a robust cluster analysis.
Differentially expressed cytokines were identified first by a paired
Student's t-test with the commonly accepted significance threshold of
p<0.05. The Student's t-test was carried out using Microsoft Excel
(Microsoft, Inc., Redman Wash.). Taking into consideration individual
variances among the patient population, a second method of selection was
also utilized.

[0528] Discriminant function analysis (DFA) (described above) was used for
selection of the set of cytokines with maximal discriminatory
capabilities between groups of samples from patients and unaffected
controls. The inventors used variant DFA named the Forward Stepwise
Analysis.

[0529] 4. Results

[0530] The data presented herein indicate the contribution of different
cytokines in controlling inflammation and also emphasize the necessity to
re-evaluate the role of the immune pathways in the pathogenesis of CD.
Immune deviation is now widely accepted as a concept in explaining the
pathogenesis of autoimmune diseases.

[0531] Crohn's Disease (CD) is a chronic systemic inflammatory rheumatic
disorder of the colon with or without extracolonic manifestations.
Mechanisms underlying the phenotypic heterogeneity of Crohn's Disease are
not known. To explore whether a molecular diagnostic characteristic
exists, serum samples from 9 patients with clinically diagnosed CD were
examined and the concentration of cytokines were quantified.

[0533] A distinct profile of cytokines was produced from patients with
Rheumatoid Arthritis (RA). This distinct cytokine profile was determined
by sampling serum for the presence of cytokines. Patients were found to
have predictive molecular cytokine profiles based on clinical disease
phenotype and disease severity. Specifically, cytokines were found to be
similar within the specific disease classifications, but the levels of
cytokines were somewhat heterogeneous with regard to individual patient,
raising the possibility that various stages of disease and disease
severity may be distinguished by this molecular diagnostic mechanism.

[0539] The clinical presentations of the cytokine profiles in the defined
patient subsets were compared by using a standard Student's t-test,
stepwise discriminate function tests, and a robust cluster analysis.
Differentially expressed cytokines were identified first by a paired
Student's t-test with the commonly accepted significance threshold of
p<0.05. The Student's t-test was carried out using Microsoft Excel
(Microsoft, Inc., Redman Wash.). Taking into consideration individual
variances among the patient population, a second method of selection was
also utilized.

Discriminant function analysis (DFA) (described above) was used for
selection of the set of cytokines with maximal discriminatory
capabilities between groups of samples from patients and unaffected
controls. The inventors used variant DFA named the Forward Stepwise
Analysis.

[0540] 4. Results.

[0541] The data presented herein indicate the contribution of different
cytokines in controlling inflammation and also emphasize the necessity to
re-evaluate the role of the immune pathways in the pathogenesis of RA.
Immune deviation is now widely accepted as a concept in explaining the
pathogenesis of autoimmune diseases.

[0542] Rheumatoid arthritis is a chronic systemic inflammatory rheumatic
disorder with or without extraskeletal manifestations. Mechanisms
underlying the phenotypic heterogeneity of rheumatoid arthritis are not
known. To explore whether a molecular diagnostic characteristic exists,
serum samples from 29 patients with clinically diagnosed RA were examined
and the concentration of cytokines were quantified.

[0544] A distinct profile of cytokines was produced from patients with
systemic lupus erythematosus (SLE). This distinct cytokine profile was
determined by sampling serum for the presence of cytokines. Patients were
found to have predictive molecular cytokine profiles based on clinical
disease phenotype and disease severity. Specifically, cytokines were
found to be similar within the specific disease classifications, but the
levels of cytokines were somewhat heterogeneous with regard to individual
patient, raising the possibility that various stages of disease and
disease severity may be distinguished by this molecular diagnostic
mechanism.

[0545] 1. Study Population

[0546] Peripheral blood serum from twenty-two patients with SLE and twelve
normal controls were used in this study. Among the SLE samples, 5 had
anti-Ro 60 and anti-La antibodies, 4 had anti-Sm and anti-nRNP
antibodies, 4 had anti-P antibodies, 3 had anti-dsDNA antibodies and 6
had anti-APL antibodies. Two SLE patients, one who developed anti-Ro over
time and another who had autoantibodies at the time of SLE diagnosis,
were observed longitudinally for a period of 13 years.

[0550] The clinical presentations of the cytokine profiles in the defined
patient subsets were compared by using a standard Student's t-test,
stepwise discriminate function tests, and a robust cluster analysis.
Differentially expressed cytokines were identified first by a paired
Student's t-test with the commonly accepted significance threshold of
p<0.05. The Student's t-test was carried out using Microsoft Excel
(Microsoft, Inc., Redman Wash.). Taking into consideration individual
variances among the patient population, a second method of selection was
also utilized.

[0551] Discriminant function analysis (DFA) (described above) was used for
selection of the set of cytokines with maximal discriminatory
capabilities between groups of samples from patients and unaffected
controls. We used variant DFA named the Forward Stepwise Analysis.

[0552] 4. Results

[0553] The data presented herein indicate the contribution of different
cytokines in controlling inflammation and also emphasize the necessity to
re-evaluate the role of the immune pathways in the pathogenesis of SLE.
Immune deviation is now widely accepted as a concept in explaining the
pathogenesis of autoimmune diseases.

[0554] Systemic Lupus Erythematosus (SLE) is a chronic systemic
inflammatory rheumatic disorder with or without major organ involvement.
Mechanisms underlying the phenotypic heterogeneity of SLE are not known.
To explore whether a molecular diagnostic characteristic exists, serum
samples from 22 patients with clinically diagnosed SLE were examined and
the concentration of cytokines were quantified.

[0555] In all specimens IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL
10, IL-12(p70), IL-13, IL-17, IFN-γ, TNF-α, GM-CSF, G-CSF,
MCP-1, and MIP-1β were analyzed using a comprehensive biometric
multiplex cytokine assay. The various subsets of SLE shared a distinctive
cytokine profile compared to normal controls. Values above the mean
control values plus three standard deviations for each cytokine were
considered as elevated. Patients with anti-Ro 60 and anti-La had elevated
levels of all the cytokines studied, except for IL-5, IL-12 and
MIP-1β, with IFN-γ increasing to 245-fold. Patients with APL
antibodies had increased levels of all cytokines studied, except for
IL-5, IL-7, IL-12 and G-CSF. Patients with anti-dsDNA antibodies, anti-P
antibodies or anti-Sm/RNP antibodies had elevated levels of IL-8, IL-10,
GM-CSF, IFN-γ and MCP-1. In the patient who developed anti-Ro under
longitudinal observation, it was observed that the cytokines IL-2, IL-6,
IL-8 and IFN-γ spiked when the patient developed anti-Ro. However,
IL-17 and MCP-1 was found to be elevated almost through the entire period
of observation in this patient while G-CSF dropped after the initial 7
years to normal levels. On the contrary another patient who had
autoantibodies at the time of SLE diagnosis had all cytokines elevated,
except for IL-10, GM-CSF, IL-5, IL-7, IL-12, IL-13 and MIP-1β for an
initial period of 7 years after which levels dropped to normal.

[0556] Pro-inflammatory cytokines and the anti-inflammatory cytokine IL-10
were elevated in all subsets of lupus patients. Only cytokines IL-2,
IL-4, IL-6, IFN-γ, and MCP-1 had markedly elevated values in
patients with anti-Ro 60 and anti-La antibodies compared to other
subsets. Anti-Ro subset had the highest IL-6 and IFN-γ levels.
IL-8, IL-17 and MCP-1 were high in all lupus subsets studied. However,
IL-5 and IL-12 were low in all lupus subsets. Both Th1 and Th2
type responses were observed in each sub-set.

Example 9

Distinct Cytokine Patterns in Familial Mediterranean Fever (FMF)

[0557] A distinct profile of cytokines was produced from patients with
Familial Mediterranean

[0558] Fever (FMF). This distinct cytokine profile was determined by
sampling serum for the presence of cytokines. Patients were found to have
predictive molecular cytokine profiles based on clinical disease
phenotype and disease severity. Specifically, cytokines were found to be
similar within the specific disease classifications, but the levels of
cytokines were somewhat heterogeneous with regard to individual patient,
raising the possibility that various stages of disease and disease
severity may be distinguished by this molecular diagnostic mechanism.

[0564] The clinical presentations of the cytokine profiles in the defined
patient subsets were compared by using a standard Student's t-test,
stepwise discriminate function tests, and a robust cluster analysis.
Differentially expressed cytokines were identified first by a paired
Student's t-test with the commonly accepted significance threshold of
p<0.05. The Student's t-test was carried out using Microsoft Excel
(Microsoft, Inc., Redman Wash.). Taking into consideration individual
variances among the patient population, a second method of selection was
also utilized.

[0565] Discriminant function analysis (DFA) (described above) was used for
selection of the set of cytokines with maximal discriminatory
capabilities between groups of samples from patients and unaffected
controls. We used variant DFA named the Forward Stepwise Analysis.

[0566] 4. Results

[0567] The data presented herein indicate the contribution of different
cytokines in controlling inflammation and also emphasize the necessity to
re-evaluate the role of the immune pathways in the pathogenesis of FMF.
Immune deviation is now widely accepted as a concept in explaining the
pathogenesis of autoimmune diseases.

[0568] Familial Mediterranean Fever is an inherited disorder usually
characterized by recurrent episodes of fever and peritonitis
(inflammation of the abdominal membrane). In 1997, researchers identified
the gene for FMF and found several different gene mutations that cause
this inherited rheumatic disease. The gene, found on chromosome 16, codes
for a protein that is found almost exclusively in granulocytes--white
blood cells important in the immune response. The protein is likely to
normally assist in keeping inflammation under control by deactivating the
immune response--without this `brake,` an inappropriate full-blown
inflammatory reaction occurs: an attack of FMF. To explore whether a
molecular diagnostic cytokine characteristic exists, serum samples from
five patients with clinically diagnosed FMF were examined and the
concentration of cytokines were quantified.

[0570] A distinct profile of cytokines was produced from patients with
Amyotrophic Lateral Sclerosis (ALS). This distinct cytokine profile was
determined by sampling serum for the presence of cytokines. Patients were
found to have predictive molecular cytokine profiles based on clinical
disease phenotype and disease severity. Specifically, cytokines were
found to be similar within the specific disease classifications, but the
levels of cytokines were somewhat heterogeneous with regard to individual
patient, raising the possibility that various stages of disease and
disease severity may be distinguished by this molecular diagnostic
mechanism.

[0571] 1. Study Population

[0572] A multiplex suspension array technology was used to quantify 28
cytokines and chemokines in archived CSF from 15 patients with
intermediate stage ALS, and from 15 age-matched patients who were not
suffering from known neurological disease.

[0575] The clinical presentations of the cytokine profiles in the defined
patient subsets were compared by using Student's t-test, stepwise
discriminate function tests, and a robust cluster analysis.
Differentially expressed cytokines were identified first by a paired
Student's t-test with the commonly accepted significance threshold of
p<0.05. The Student's t-test was carried out using Microsoft Excel
(Microsoft, Inc., Redman Wash.). Taking into consideration individual
variances among the patient population, a second method of selection was
also utilized.

[0576] Discriminant function analysis (DFA) (described above) was used for
selection of the set of cytokines with maximal discriminatory
capabilities between groups of samples from patients and unaffected
controls. We used variant DFA named the Forward Stepwise Analysis.

[0577] 4. Results

[0578] The data presented herein indicate the contribution of different
cytokines in controlling inflammation and also emphasize the necessity to
re-evaluate the role of the immune pathways in the pathogenesis of ALS.
Immune deviation is now widely accepted as a concept in explaining the
pathogenesis of autoimmune diseases.

[0579] The neuroinflammatory hypothesis contends that dysregulated
cytokine networks contribute to neuropathology in amyotrophic lateral
sclerosis (ALS) and other neurodegenerative diseases. Recent findings
indicate that cytokine expression changes correlate with disease onset
and progression in mouse models of familial amyotrophic lateral sclerosis
(ALS). A study was undertaken to determine whether similar cytokine
alterations could be observed in cerebrospinal fluid (CSF) obtained from
humans with ALS. A multiplex suspension array technology was used to
quantify 17 cytokines and chemokines in archived CSF from 15 patients
with intermediate stage ALS, and from 15 age-matched patients who were
not suffering from known neurological disease.

[0580] Additionally, enzyme-linked immunosorbent assays (ELISAs) were
employed to measure inflammatory molecules prostaglandin E2
(PGE2), leukotriene B4 (LTB4) and the apoptosis biomarker
cleaved tau (c-tau) in the same CSF samples. Mean levels of all analytes
except IL-12 were increased in the ALS group relative to non-afflicted
subjects; mean IL-12 decreased by 50% in the ALS group. Macrophage
inflammatory protein 1-beta (MIP-1β was significantly increased at
the 95% confidence level while IL-13 and IL-8 were significant at the 90%
level. As a fraction of the total cytokine pool, IL-13 concentrations
tended to be elevated (p<0.07) and IL-12 were decreased (p<0.08) in
the ALS group. Consequently, the ratio of IL-13/IL-12 increased 4-fold in
ALS (p<0.01). These data suggest an alteration in the Th1/Th2 cytokine
blance, and suggest that multiplex cytokine suspension arrays may be
useful for indexing neurological disease in human clinical studies.

[0581] The results strongly suggest a broad-spectrum dysregulation of
cytokine components in the intermediate stages of human sporadic ALS. In
particular, four proteins (MIP-1β, IL-8, IL-12p70 and IL-13) are
especially notable analytes that differ between ALS and non-diseased
cerebrospinal fluid. These findings begin to corroborate the
neuroinflammatory hypothesis of ALS in the human disease.

Example 11

Distinct Cytokine Profiles in Sjogren's Syndrome (SS)

[0582] A distinct profile of cytokines was produced from patients with
Sjogren's Syndrome (SS) This distinct cytokine profile was determined by
sampling serum for the presence of cytokines. Patients were found to have
predictive molecular cytokine profiles based on clinical disease
phenotype and disease severity. Specifically, cytokines were found to be
similar within the specific disease classifications, but the levels of
cytokines were somewhat heterogeneous with regard to individual patient,
raising the possibility that various stages of disease and disease
severity may be distinguished by this molecular diagnostic mechanism.

[0583] 1. Study Population

[0584] A multiplex suspension array technology was used to quantify 28
cytokines and chemokines in serum and plasma of 11 patients who fulfilled
the revised European classification criteria for primary SS, and healthy
controls.

[0588] The clinical presentations of the cytokine profiles in the defined
patient subsets were compared by using Student's t-test, stepwise
discriminate function tests, and a robust cluster analysis.
Differentially expressed cytokines were identified first by a paired
Student's t-test with the commonly accepted significance threshold of
p<0.05. The Student's t-test was carried out using Microsoft Excel
(Microsoft, Inc., Redman Wash.). Taking into consideration individual
variances among the patient population, a second method of selection was
also utilized.

[0589] Discriminant function analysis (DFA) (described above) was used for
selection of the set of cytokines with maximal discriminatory
capabilities between groups of samples from patients and unaffected
controls. We used variant DFA named the Forward Stepwise Analysis.

[0590] 4. Results

[0591] The key cytokines/chemokines that discriminate SSGC+ patients from
GC- patients and controls are BAFF, CCL2 (MCP-1), IL-12p40, CXCL8 (IL-8)
and CCL11 (Eotaxin). We further demonstrated the dominant discriminative
role of BAFF and CCL2 (MCP-1) between SSGC+ and SSGC- and signified its
important role in ectopic salivary GC development in SS. The role of BAFF
in GC formation in animal models has been investigates and showed normal
induction but attenuated progression of GC responses in BAFF and BAFF-R
signaling-deficient mice (14). Our results indicate that BAFF has a
crucial `driving force` in the development of ectopic GCs, a potential
pre-stage of lymphomagenesis characterized by B-cell hyperactivity,
proliferation and autoantibody secretion. Interestingly, patients with
B-cell non-Hodgkin's lymphoma (NEIL) have elevated BAFF serum levels and
in positive correlation with disease-severity. Also, BAFF levels
correlate with response to therapy in patients with B-cell NHL.
Responding patients had a significantly lower BAFF level than those with
progressive disease (42). Considering these findings, we hypothesize that
in SS, elevated levels of BAFF, together with a unique pattern of
elevated circulating cytokines, such as CCL2 (MCP-1), IL-12p40, CXCL8
(IL-8) and CCL11 (Eotaxin) among other factors, lead to GC formation and
presumably this chronic stimulation might lead to B-cell neoplastic
transformation. Moreover, since BAFF is produced by macrophages,
monocytes and dendritic cells (43-45), the discriminant potential of CCL2
(MCP-1) is presumably by stimulating monocytes/macrophages to produce
BAFF, therefore indirectly support the BAFF-mediated processes.

The key cytokines/chemokines that discriminate SSGC+ patients from GC-
patients and controls are BAFF, CCL2 (MCP-1), IL-12p40, CXCL8 (IL-8) and
CCL11 (Eotaxin). We further demonstrated the dominant discriminative role
of BAFF and CCL2 (MCP-1) between SSGC+ and SSGC- and signified its
important role in ectopic salivary GC development in SS. The role of BAFF
in GC formation in animal models has been investigates and showed normal
induction but attenuated progression of GC responses in BAFF and BAFF-R
signaling-deficient mice (14). Our results indicate that BAFF has a
crucial `driving force` in the development of ectopic GCs, a potential
pre-stage of lymphomagenesis characterized by B-cell hyperactivity,
proliferation and autoantibody secretion. Interestingly, patients with
B-cell non-Hodgkin's lymphoma (NHL) have elevated BAFF serum levels and
in positive correlation with disease-severity. Also, BAFF levels
correlate with response to therapy in patients with B-cell NHL.
Responding patients had a significantly lower BAFF level than those with
progressive disease (42). Considering these findings, we hypothesize that
in SS, elevated levels of BAFF, together with a unique pattern of
elevated circulating cytokines, such as CCL2 (MCP-1), IL-12p40, CXCL8
(IL-8) and CCL11 (Eotaxin) among other factors, lead to GC formation and
presumably this chronic stimulation might lead to B-cell neoplastic
transformation. Moreover, since BAFF is produced by macrophages,
monocytes and dendritic cells (43-45), the discriminant potential of CCL2
(MCP-1) is presumably by stimulating monocytes/macrophages to produce
BAFF, therefore indirectly support the BAFF-mediated processes. Our
results imply that a complex disorder of circulating serum cytokines
contribute to Sjogren's Syndromes. We believe that the utilization of the
multiplex cytokine array system in SS provides a powerful tool to
sub-categorize the disease and find the relevant features of
heterogeneous patients and along with common clinical and laboratory
parameters help to evaluate these two types of the disease.

[0592] The key cytokines/chemokines that discriminate patients and
controls are CCL2/MCP-1, IL-12, CXCL8/IL-8, CCL11/Eotaxin, TNFα,
IL-2, IFNα, IL-15, IL17, IL-1β, IL-6, and GM-CSF. In separate
ongoing studies utilizing the multiplex cytokine assay, serial serum
cytokine level measurements did not fluctuate significantly. Moreover, to
exclude modulations in the circulating cytokine levels we selected
patients in this study, who at the time of the analysis were not taking
immunmodulatory medications, which would seriously affect the cytokine
levels.

[0593] The inventors conclude that the multiplex cytokine assay along with
the utilization of DFA can clearly distinguish SS patients into distinct
subsets and from healthy controls. This information represents a powerful
tool to pinpoint candidate discriminative cytokines on the basis of which
the follow-up of patients can be carried out. The identification of
cytokine-markers of the potentially prelymphomatous disease entity can be
important not only in diagnosis, but also in designing selective
anti-cytokine therapies that would inhibit the perpetuation of ectopic
germinal center formation commonly found in Sjogren's Syndrome.

Example 12

Distinct Cytokine Profiles in Early Arthritis

[0594] A distinct profile of cytokines was produced from patients with
Early Arthritis. This distinct cytokine profile was determined by
sampling serum for the presence of cytokines. Patients were found to have
predictive molecular cytokine profiles based on clinical disease
phenotype and disease severity. Specifically, cytokines were found to be
similar within the specific disease classifications, but the levels of
cytokines were somewhat heterogeneous with regard to individual patient,
raising the possibility that various stages of disease and disease
severity may be distinguished by this molecular diagnostic mechanism.

[0595] 1. Study Population

[0596] A multiplex suspension array technology was used to quantify 28
cytokines and chemokines in plasma from 41 patients with early and
undifferentiated arthritis and 21 healthy controls.

[0600] The clinical presentations of the cytokine profiles in the defined
patient subsets were compared by using Student's t-test, stepwise
discriminate function tests, and a robust cluster analysis.
Differentially expressed cytokines were identified first by a paired
Student's t-test with the commonly accepted significance threshold of
p<0.05. The Student's t-test was carried out using Microsoft Excel
(Microsoft, Inc., Redman Wash.). Taking into consideration individual
variances among the patient population, a second method of selection was
also utilized.

[0601] Discriminant function analysis (DFA) (described above) was used for
selection of the set of cytokines with maximal discriminatory
capabilities between groups of samples from patients and unaffected
controls. The inventors used variant DFA named the Forward Stepwise
Analysis.

[0602] 4. Results

[0603] Both pro- and anti-inflammatory cytokines were elevated in patients
over controls. Ther cytokines upregulated in patients included
CCL4/MIP1β, CXCL8/IL-8, IL-2, IL-12, IL-17, IL-13, TNFα, IL-4,
IL-5, and IL-10. Early untreated inflammatory arthritis can be
categorized into distinct subgroups based on cytokine profiles.
Integration of cytokine profiles may assist prognostication and treatment
decisions in these patients.

Example 13

Distinct Cytokine Profiles in Psoriasis (PSO)

[0604] A distinct profile of cytokines was produced from patients with
Psoriasis (PSO). This distinct cytokine profile was determined by
sampling serum for the presence of cytokines. Patients were found to have
predictive molecular cytokine profiles based on clinical disease
phenotype and disease severity. Specifically, cytokines were found to be
similar within the specific disease classifications, but the levels of
cytokines were somewhat heterogeneous with regard to individual patient,
raising the possibility that various stages of disease and disease
severity may be distinguished by this molecular diagnostic mechanism.

[0605] J. Study Population

[0606] A multiplex suspension array technology was used to quantify 28
cytokines and chemokines in serum 5 patients with clinically diagnosed
psoriasis and 5 healthy controls.

[0610] The clinical presentations of the cytokine profiles in the defined
patient subsets were compared by using Student's t-test, stepwise
discriminate function tests, and a robust cluster analysis.
Differentially expressed cytokines were identified first by a paired
Student's t-test with the commonly accepted significance threshold of
p<0.05. The Student's t-test was carried out using Microsoft Excel
(Microsoft, Inc., Redman Wash.). Taking into consideration individual
variances among the patient population, a second method of selection was
also utilized.

[0611] Discriminant function analysis (DFA) (described above) was used for
selection of the set of cytokines with maximal discriminatory
capabilities between groups of samples from patients and unaffected
controls. We used variant DFA named the Forward Stepwise Analysis.

[0612] 4. Results

[0613] Serum cytokines IL-1β, IL-2, CXCL8/IL-8, IL-13, G-CSF, and
CCL4/MIP-1β were upregulated in psoriasis patients. FGF was the down
regulated to levels lower than that seen in controls. The series of serum
cytokines in this profile is unique to psoriasis The identification of
cytokine-markers in psoriasis can be important not only in diagnosis, but
also in designing selective anti-cytokine therapies that would prevent
the scaring and physical discomfort of the disease.